Biomarkers to identify patients that will respond to treatment and treating such patients

ABSTRACT

The invention relates to methods for using biomarkers to identify cancer patients that will or are likely to respond to treatment. Specifically, the invention relates to the use of one or more of three association studies of cancer types, gene mutations, or gene expression levels in order to identify cancer patients that will or are likely to respond to treatment with a histone deacetylase (HDAC) inhibitor, alone or in combination with another cancer treatment. The methods may, optionally, further include treating such patient with an HDAC inhibitor, alone or in combination with another cancer treatment.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/778,260, filed Mar. 12, 2013; U.S. Provisional Application No.61/664,471, filed Jun. 26, 2012; and U.S. Provisional Application No.61/635,336, filed Apr. 19, 2012. The contents of each of theseapplications are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

Provided herein are methods for using biomarkers to identify cancerpatients who will or are likely to respond to treatment. Specifically,the methods relate to the use of one or more of three associationstudies of cancer types, gene mutations, or gene expression levels inorder to identify cancer patients who will or are likely to respond totreatment with a histone deacetylase (HDAC) inhibitor, alone or incombination with another cancer treatment. The methods can, optionally,further include treating such patients with an HDAC inhibitor, alone orin combination with another cancer treatment. Additional methods anduses for these biomarkers and association studies are also disclosed.

BACKGROUND OF THE INVENTION

Cancer is one of the leading causes of death in the United States and inthe world. It is estimated that approximately 1.6 million new cases ofcancer will occur in the United States in 2012. It is also estimatedthat approximately 575,000 people will die from cancer in the UnitedStates in 2012.

Cancer grows out of normal cells in the body. Normal cells multiply whenthe body needs them, and die when the body doesn't need them. Canceroccurs when the cells in the body grow and multiply out of control.There are many different types of cancer, which can develop in almostany organ or tissue in the body.

There are many causes of cancer, such as exposure to carcinogenicchemicals, use of tobacco, drinking excess alcohol, exposure toenvironmental toxins, exposure to excessive sunlight, genetic problems,obesity, radiation, and viruses. In addition, the cause of many cancersremains unknown.

The symptoms of cancer depend on the type and location of the cancer.

Most cancers are diagnosed by biopsy. Depending on the location of thetumor, the biopsy may be a simple procedure or a serious operation.

Treatment varies based on the type of cancer, its stage of progression,and whether it has spread to other parts of the body. Treatment optionsinclude surgery to remove the cancer, radiation therapy (the use ofhigh-powered x-rays, particles, or radioactive seeds to kill the cancercells), chemotherapy (the use of drugs to kill the cancer cells), or acombination of the above.

Cancer therapy has many side effects. For example, radiation therapy ismost harmful to rapidly growing cells, such as cancer cells, andspecifically damages the DNA of cancer cells. However, radiation therapyalso affects normal cells. People that receive radiation therapy oftenhave hair loss; skin pain; red, burning skin; shedding of the outerlayer of the skin; increased skin coloring; thinning of skin tissue;itching; fatigue and malaise; low blood counts; difficulty or painfulswallowing; edema; changes in taste; loss of appetite; nausea; vomiting;and increased susceptibility to infection. Similar to radiation therapy,chemotherapy affects cells that divide often, such as cancer cells.However, chemotherapy also affects normal cells, such as those found inthe blood, hair, and lining of the gastrointestinal tract. People thatreceive chemotherapy are more likely to have infections; become tiredmore easily; bleed too much; feel pain from damage to the nerves; have adry mouth, mouth sores, or swelling of the mouth; have a poor appetiteand lose weight; and have upset stomach, vomiting, and diarrhea.

Accordingly, there is a need to quickly and reliably choose a course ofcancer treatment that will achieve the best outcome for the personreceiving treatment while exposing the person to the least amount ofchemotherapy drugs as possible, which will keep side effects to aminimum.

SUMMARY OF THE INVENTION

To meet this and other needs, provided herein are methods for usingbiomarkers to identify cancer patients who will or are likely to respondto treatment. Specifically, the methods relate to the use of one or moreof three association studies of cancer types, gene mutations, or geneexpression levels in order to identify cancer patients who will or arelikely to respond to treatment with a histone deacetylase (HDAC)inhibitor, alone or in combination with another cancer treatment,therapy or drug.

An embodiment of the invention comprises an association study of cancertypes, which associates treatment effect by tumor type.

Another embodiment of the invention comprises an association study ofgene mutations, which associates gene mutation analysis with treatmenttype and tumor type.

A further embodiment of the invention comprises an association study ofgene expression levels, which associates gene expression analysis withtumor type.

An embodiment of the invention provides a method for using a biomarkerto identify a cancer patient who will or is likely to respond totreatment with a histone deacetylase (HDAC) inhibitor, alone or incombination with another cancer treatment, by interpreting data from oneor more of the above three association studies of cancer types, genemutations, or gene expression levels.

An embodiment of the invention provides a method for using a biomarkerto predict whether a cancer patient will or is likely to respond totreatment with a histone deacetylase (HDAC) inhibitor, alone or incombination with another cancer treatment, by interpreting data from oneor more of the above three association studies of cancer types, genemutations, or gene expression levels.

An embodiment of the invention provides a method for using a biomarkerto predict the response of a cancer patient to treatment with a histonedeacetylase (HDAC) inhibitor, alone or in combination with anothercancer treatment, by interpreting data from one or more of the abovethree association studies of cancer types, gene mutations, or geneexpression levels.

An embodiment of the invention provides a method for using a biomarkerto predict cancer cell inhibition in response to treatment with ahistone deacetylase (HDAC) inhibitor, alone or in combination withanother cancer treatment, by interpreting data from one or more of theabove three association studies of cancer types, gene mutations, or geneexpression levels.

An embodiment of the invention provides a method for using a biomarkerto predict tumor cell killing in response to treatment with a histonedeacetylase (HDAC) inhibitor, alone or in combination with anothercancer treatment, by interpreting data from one or more of the abovethree association studies of cancer types, gene mutations, or geneexpression levels.

An embodiment of the invention provides a method for using a biomarkerto predict the sensitivity of cancer cell growth to treatment with ahistone deacetylase (HDAC) inhibitor, alone or in combination withanother cancer treatment, by interpreting data from one or more of theabove three association studies of cancer types, gene mutations, or geneexpression levels.

An embodiment of the invention provides a method for using a biomarkerto predict the sensitivity of tumor cell growth to treatment with ahistone deacetylase (HDAC) inhibitor, alone or in combination withanother cancer treatment, by interpreting data from one or more of theabove three association studies of cancer types, gene mutations, or geneexpression levels.

An embodiment of the invention provides a method for using a biomarkerto identify a cancer patient who is likely to benefit from treatmentwith a histone deacetylase (HDAC) inhibitor, alone or in combinationwith another cancer treatment, by interpreting data from one or more ofthe above three association studies of cancer types, gene mutations, orgene expression levels.

An embodiment of the invention provides a method for treating cancercomprising administering a histone deacetylase (HDAC) inhibitor, aloneor in combination with another cancer treatment, to a patient who,according to data from one or more of the above three associationstudies of cancer types, gene mutations, or gene expression levels, willor is likely to respond to such treatment.

An embodiment of the invention provides a method for treating cancer ina patient comprising the steps of predicting whether a cancer patientwill respond to treatment with a histone deacetylase (HDAC) inhibitor,alone or in combination with another cancer treatment, by interpretingdata from one or more of the above three association studies of cancertypes, gene mutations, or gene expression levels, and administering tothe patient a therapeutically effective amount of a histone deacetylase(HDAC) inhibitor, alone or in combination with another cancer treatment,if it is determined that the patient will or likely to respond to suchtreatment.

An embodiment of the invention provides a method for re-treating cancerin a patient comprising the steps of predicting whether a cancer patientwill respond to treatment with a histone deacetylase (HDAC) inhibitor,alone or in combination with another cancer treatment, by interpretingdata from one or more of the above three association studies of cancertypes, gene mutations, or gene expression levels, and administering tothe patient a therapeutically effective amount of a histone deacetylase(HDAC) inhibitor, alone or in combination with another cancer treatment,if it is determined that the patient will or is likely to respond tosuch treatment.

An embodiment of the invention provides a method for modifying cancertreatment in a patient comprising the steps of predicting whether acancer patient will respond to treatment with a histone deacetylase(HDAC) inhibitor, alone or in combination with another cancer treatment,by interpreting data from one or more of the above three associationstudies of cancer types, gene mutations, or gene expression levels, andadministering to the patient a therapeutically effective amount of ahistone deacetylase (HDAC) inhibitor, alone or in combination withanother cancer treatment, if it is determined that the patient will oris likely to respond to such treatment.

An embodiment of the invention provides a method for optimizing cancertreatment in a patient comprising the steps of predicting whether acancer patient will respond to treatment with a histone deacetylase(HDAC) inhibitor, alone or in combination with another cancer treatment,by interpreting data from one or more of the above three associationstudies of cancer types, gene mutations, or gene expression levels, andadministering to the patient a therapeutically effective amount of ahistone deacetylase (HDAC) inhibitor, alone or in combination withanother cancer treatment, if it is determined that the patient will oris likely to respond to such treatment.

In a specific embodiment of the invention, the invention provides amethod for predicting whether a breast cancer patient will respond totreatment with a histone deacetylase (HDAC) inhibitor comprising thesteps of: a) determining whether the human epidermal growth factorreceptor 2 (Her2) protein is overexpressed, as compared to a normalizedprotein expression level of the protein, in a biological sample from thebreast cancer patient; and b) correlating the presence of suchoverexpression as an indication that the patient will respond to suchtreatment, or correlating the absence of such overexpression as anindication that the patient will not respond to such treatment.

In another specific embodiment of the invention, the invention providesa method for predicting whether a colorectal cancer patient will respondto combination treatment with a histone deacetylase (HDAC) inhibitor anda proteasome inhibitor comprising the steps of: a) determining whether agene mutation in the SMAD family member 4 (SMAD4) gene is present in abiological sample from the colorectal cancer patient; and b) correlatingthe presence of a gene mutation as an indication that the patient willrespond to such treatment, or correlating the absence of a gene mutationas an indication that the patient will not respond to such treatment.

In yet another specific embodiment of the invention, the inventionprovides a method for predicting whether a cancer patient will respondto combination treatment with a histone deacetylase (HDAC) inhibitor anda proteasome inhibitor comprising the steps of: a) determining whether agene mutation in a gene selected from the group consisting ofphosphatase and tensin homolog (PTEN), epidermal growth factor receptoroncogene (EGFR), histone-lysine N-methyltransferase (EZH2), SET domaincontaining 2 (SETD2), and von Hippel-Lindau tumor suppressor (VHL), ispresent in a biological sample from the cancer patient; and b)correlating the presence of one or more such mutations as an indicationthat the patient will respond to such treatment, or correlating theabsence of such mutations as an indication that the patient will notrespond to such treatment.

In another specific embodiment of the invention, the invention providesa method for predicting whether a cancer patient will respond totreatment with a histone deacetylase (HDAC) inhibitor comprising thesteps of: a) measuring the expression level of a gene selected from thegroup consisting of pterin-4 alpha-carbinolaminedehydratase/dimerization cofactor of hepatocyte nuclear factor 1 alpha(PCBD1); protein phosphatase 2, regulatory subunit B, gamma isoform(PPP2R2C); neural precursor cell expressed, developmentallydownregulated 4 (NEDD4); prolyl 4-hydroxylase, alpha polypeptide II(P4HA2); SLC2A4 regulator (SLC2A4RG); sulfatase 2 (SULF2); lysosomalprotein transmembrane 4 alpha (LAPTM4A); 3′-phosphoadenosine5′-phosphosulfate synthase 2 (PAPSS2); aldo-keto reductase family 1,member C1 (dihydrodiol dehydrogenase 1; 20-alpha(3-alpha)-hydroxysteroid dehydrogenase) (AKR1C1); protein tyrosinephosphatase, non-receptor type 12 (PTPN12); DCN1, defective in cullinneddylation 1, domain containing 4 (S. cerevisiae) (DCUN1D4);ras-related C3 botulinum toxin substrate 2 (RAC2); acyl-Coexzyme Adehydrogenase, C-4 to C-112 straight chain (ACADM); Rho GTPaseactivating protein 4 (ARHGAP4); ATPase type 13A1 (ATP13A1); chemokinereceptor 7 (CCR7); coronin 7 (CORO7); CXXC finger 4 (CXXC4);differentially expressed in FDCP 6 homolog (DEF6); KRI1 homolog (KRI1);limb region 1 homolog (LMBR1L); leukotriene B4 receptor (LTB4R);RAD54-like 2 (RAD54L2); chromosome X open reading frame 21 (CXorf21);SREBF chaperone (SCAP); selectin L (SELL); splicing factor 3a, subunit 2(SF3A2); Lyrm7 homolog (LYRM7); O-linked N-acetylglucosamine transferase(OGT); tubulin, alpha 3c (TUBA3C); tubulin, alpha 3d (TUBA3D); KH-typesplicing regulatory protein (KHSRP); DEAH (Asp-Glu-Ala-His) boxpolypeptide 30 (DHX30); APEX nuclease (apurinic/apyrimidinicendonuclease) 2 (APEX2); and abhydrolase domain containing 14A (ABHD14A)in a biological sample from the cancer patient; and b) correlating a lowexpression level, as compared to a normalized gene expression level ofthe gene, of any one or more of the genes PCBD1, PPP2R2C, NEDD4, P4HA2,SLC2A4RG, SULF2, LAPTM4A, PAPSS2, AKR1C1, PTPN12, and DCUN1D4 as anindication that the patient will respond to such treatment; orcorrelating a high expression level, as compared to a normalized geneexpression level of the gene, of any one or more of the genes RAC2,ACADM, ARHGAP4, ATP13A1, CCR7, CORO7, CXXC4, DEF6, KRI1, LMBR1L, LTB4R,RAD54L2, CXorf21, SCAP, SELL, SF3A2, LYRM7, OGT, TUBA3C, TUBA3D, KHSRP,DHX30, APEX2, and ABHD14A as an indication that the patient will respondto such treatment.

In yet another specific embodiment of the invention, the inventionprovides a method for predicting whether a cancer patient will respondto combination treatment with a histone deacetylase (HDAC) inhibitor anda proteasome inhibitor comprising the steps of: a) measuring theexpression level of a gene selected from the group consisting ofUDP-glucose dehydrogenase (UGDH); H2A histone family, member Y2(H2AFY2); myosin VC (MYO5C); nephronectin (NPNT); KIAA1598 (KIAA1598);serglycin (SRGN); collagen, type VI, alpha 3 (COL6A3); G-proteinsignaling modulator 3 (GPSM3); hydroxysteroid dehydrogenase 1 (HSD11B1);peroxisomal biogenesis factor 6 (PEX6); ras-related C3 botulinum toxinsubstrate 2 (RAC2); synovial sarcoma, X breakpoint 5 (SSX5); andacyl-Coenzyme A binding domain containing 3 (ACBD3); in a biologicalsample from the cancer patient; and b) correlating a low expressionlevel, as compared to a normalized gene expression level of the gene, ofany one or more of the genes UGDH, H2AFY2, MYO5C, NPNT, and KIAA1598 asan indication that the patient will respond to such treatment; orcorrelating a high expression level, as compared to a normalized geneexpression level of the gene, of any one or more of the genes SRGN,COL6A3, GPSM3, HSD11B1, PEX6, RAC2, SSX5, and ACBD3 as an indicationthat the patient will respond to such treatment.

In a further specific embodiment of the invention, the inventionprovides a method for predicting whether a cancer patient will respondto treatment with a histone deacetylase (HDAC) inhibitor, alone or incombination with a proteasome inhibitor, comprising the steps of: a)evaluating the data from one or more association studies comprising: 1)an association study that associates treatment effect by tumor type,wherein brain/neuron cancer, breast cancer, lymphoid cancer, kidneycancer, colon/large intestine cancer, and skin cancer are determined tobe sensitive to combination treatment with a histone deacetylaseinhibitor and a proteasome inhibitor, 2) an association study thatassociates gene mutation analysis with treatment type and tumor type,wherein i) the overexpression of the human epidermal growth factorreceptor 2 (Her2) protein, as compared to a normalized proteinexpression level of the protein, in a biological sample from a breastcancer patient is an indication that the patient will respond totreatment with a histone deacetylase inhibitor, ii) the presence of agene mutation in the SMAD family member 4 (SMAD4) gene in a biologicalsample from a colorectal cancer patient is an indication that thepatient will respond to combination treatment with a histone deacetylaseinhibitor and a proteasome inhibitor, or iii) the presence of one ormore gene mutations in a gene selected from the group consisting ofphosphatase and tensin homolog (PTEN), epidermal growth factor receptoroncogene (EGFR), histone-lysine N-methyltransferase (EZH2), SET domaincontaining 2 (SETD2), and von Hippel-Lindau tumor suppressor (VHL) in abiological sample from a cancer patient is an indication that thepatient will respond to combination treatment with a histone deacetylaseinhibitor and a proteasome inhibitor, or 3) an association study thatassociates gene expression analysis with treatment type, comprising i)measuring the expression level of a gene selected from the groupconsisting of pterin-4 alpha-carbinolamine dehydratase/dimerizationcofactor of hepatocyte nuclear factor 1 alpha (PCBD1); proteinphosphatase 2, regulatory subunit B, gamma isoform (PPP2R2C); neuralprecursor cell expressed, developmentally down-regulated 4 (NEDD4);prolyl 4-hydroxylase, alpha polypeptide II (P4HA2); SLC2A4 regulator(SLC2A4RG); sulfatase 2 (SULF2); lysosomal protein transmembrane 4 alpha(LAPTM4A); 3′-phosphoadenosine 5′-phosphosulfate synthase 2 (PAPSS2);aldo-keto reductase family 1, member C1 (dihydrodiol dehydrogenase 1;20-alpha (3-alpha)-hydroxysteroid dehydrogenase) (AKR1C1); proteintyrosine phosphatase, non-receptor type 12 (PTPN12); DCN1, defective incullin neddylation 1, domain containing 4 (S. cerevisiae) (DCUN1D4);ras-related C3 botulinum toxin substrate 2 (RAC2); acyl-Coexzyme Adehydrogenase, C-4 to C-112 straight chain (ACADM); Rho GTPaseactivating protein 4 (ARHGAP4); ATPase type 13A1 (ATP13A1); chemokinereceptor 7 (CCR7); coronin 7 (CORO7); CXXC finger 4 (CXXC4);differentially expressed in FDCP 6 homolog (DEF6); KRI1 homolog (KRI1);limb region 1 homolog (LMBR1L); leukotriene B4 receptor (LTB4R);RAD54-like 2 (RAD54L2); chromosome X open reading frame 21 (CXorf21);SREBF chaperone (SCAP); selectin L (SELL); splicing factor 3a, subunit 2(SF3A2); Lyrm7 homolog (LYRM7); O-linked N-acetylglucosamine transferase(OGT); tubulin, alpha 3c (TUBA3C); tubulin, alpha 3d (TUBA3D); KH-typesplicing regulatory protein (KHSRP); DEAH (Asp-Glu-Ala-His) boxpolypeptide 30 (DHX30); APEX nuclease (apurinic/apyrimidinicendonuclease) 2 (APEX2); and abhydrolase domain containing 14A (ABHD14A)in a biological sample from the cancer patient; and a) correlating a lowexpression level, as compared to a normalized gene expression level ofthe gene, of any one or more of the genes PCBD1, PPP2R2C, NEDD4, P4HA2,SLC2A4RG, SULF2, LAPTM4A, PAPSS2, AKR1C1, PTPN12, and DCUN1D4 as anindication that the patient will respond to treatment with a histonedeacetylase inhibitor; or correlating a high expression level, ascompared to a normalized gene expression level of the gene, of any oneor more of the genes RAC2, ACADM, ARHGAP4, ATP13A1, CCR7, CORO7, CXXC4,DEF6, KRI1, LMBR1L, LTB4R, RAD54L2, CXorf21, SCAP, SELL, SF3A2, LYRM7,OGT, TUBA3C, TUBA3D, KHSRP, DHX30, APEX2, and ABHD14A as an indicationthat the patient will respond to treatment with a histone deacetylaseinhibitor, or ii) measuring the expression level of a gene selected fromthe group consisting of UDP-glucose dehydrogenase (UGDH); H2A histonefamily, member Y2 (H2AFY2); myosin VC (MYO5C); nephronectin (NPNT);KIAA1598 (KIAA1598); serglycin (SRGN); collagen, type VI, alpha 3(COL6A3); G-protein signaling modulator 3 (GPSM3); hydroxysteroiddehydrogenase 1 (HSD11B1); peroxisomal biogenesis factor 6 (PEX6);ras-related C3 botulinum toxin substrate 2 (RAC2); synovial sarcoma, Xbreakpoint 5 (SSX5); and acyl-Coenzyme A binding domain containing 3(ACBD3); in a biological sample from the cancer patient; and a)correlating a low expression level, as compared to a normalized geneexpression level of the gene, of any one or more of the genes UGDH,H2AFY2, MYO5C, NPNT, and KIAA1598 as an indication that the patient willrespond to combination treatment with a histone deacetylase inhibitorand a proteasome inhibitor; or correlating a high expression level, ascompared to a normalized gene expression level of the gene, of any oneor more of the genes SRGN, COL6A3, GPSM3, HSD11B1, PEX6, RAC2, SSX5, andACBD3 as an indication that the patient will respond to combinationtreatment with a histone deacetylase inhibitor and a proteasomeinhibitor, and b) evaluating the data regarding cancer types, genemutations, or gene expression levels to determine whether the patientwill respond to such treatment.

In preferred specific embodiments of the invention, the cancer isselected from the group consisting of: brain/neuronal cancer, breastcancer, cancer of the central nervous system, haematopoietic andlymphoid tissue cancer, kidney cancer, cancer of the large intestine,liver cancer, lung cancer, cancer of the oesophagus, pancreatic cancer,prostate cancer, skin cancer, soft tissue cancer, and stomach cancer.

In preferred specific embodiments of the invention, the HDAC inhibitoris a HDAC6 inhibitor. In more preferred specific embodiments of theinvention, the HDAC6 inhibitor is a compound of formula I:

or a pharmaceutically acceptable salt, ester or prodrug thereof. In mostpreferred specific embodiments of the invention, the compound of formulaI is

which is referred to herein as Compound A.

In preferred specific embodiments of the invention, the proteasomeinhibitor is bortezomib.

In preferred specific embodiments of the invention, the biologicalsample is a biopsy sample.

In preferred specific embodiments of the invention, the methods furthercomprise the step of administering to the patient a therapeuticallyeffective amount of a HDAC inhibitor. In alternative preferred specificembodiments of the invention, the methods further comprise the step ofadministering to the patient a therapeutically effective amount of aHDAC inhibitor and a proteasome inhibitor.

A preferred embodiment of the invention provides a kit comprising anucleic acid that hybridizes under stringent conditions with any one ofthe genes selected from the group consisting of erythroblastic leukemiaviral oncogene homolog 2 (ERBB2); SMAD family member 4 (SMAD4);phosphatase and tensin homolog (PTEN); epidermal growth factor receptoroncogene (EGFR); histone-lysine N-methyltransferase (EZH2); SET domaincontaining 2 (SETD2); von Hippel-Lindau tumor suppressor (VHL); pterin-4alpha-carbinolamine dehydratase/dimerization cofactor of hepatocytenuclear factor 1 alpha (PCBD1); protein phosphatase 2, regulatorysubunit B, gamma isoform (PPP2R2C); neural precursor cell expressed,developmentally downregulated 4 (NEDD4); prolyl 4-hydroxylase, alphapolypeptide II (P4HA2); SLC2A4 regulator (SLC2A4RG); sulfatase 2(SULF2); lysosomal protein transmembrane 4 alpha (LAPTM4A);3′-phosphoadenosine 5′-phosphosulfate synthase 2 (PAPSS2); aldo-ketoreductase family 1, member C1 (dihydrodiol dehydrogenase 1; 20-alpha(3-alpha)-hydroxysteroid dehydrogenase) (AKR1C1); protein tyrosinephosphatase, non-receptor type 12 (PTPN12); DCN1, defective in cullinneddylation 1, domain containing 4 (S. cerevisiae) (DCUN1D4);ras-related C3 botulinum toxin substrate 2 (RAC2); acyl-Coexzyme Adehydrogenase, C-4 to C-112 straight chain (ACADM); Rho GTPaseactivating protein 4 (ARHGAP4); ATPase type 13A1 (ATP13A1); chemokinereceptor 7 (CCR7); coronin 7 (CORO7); CXXC finger 4 (CXXC4);differentially expressed in FDCP 6 homolog (DEF6); KRI1 homolog (KRI1);limb region 1 homolog (LMBR1L); leukotriene B4 receptor (LTB4R);RAD54-like 2 (RAD54L2); chromosome X open reading frame 21 (CXorf21);SREBF chaperone (SCAP); selectin L (SELL); splicing factor 3a, subunit 2(SF3A2); Lyrm7 homolog (LYRM7); O-linked N-acetylglucosamine transferase(OGT); tubulin, alpha 3c (TUBA3C); tubulin, alpha 3d (TUBA3D); KH-typesplicing regulatory protein (KHSRP); DEAH (Asp-Glu-Ala-His) boxpolypeptide 30 (DHX30); APEX nuclease (apurinic/apyrimidinicendonuclease) 2 (APEX2); abhydrolase domain containing 14A (ABHD14A);UDP-glucose dehydrogenase (UGDH); H2A histone family, member Y2(H2AFY2); myosin VC (MYO5C); nephronectin (NPNT); KIAA1598 (KIAA1598);serglycin (SRGN); collagen, type VI, alpha 3 (COL6A3); G-proteinsignaling modulator 3 (GPSM3); hydroxysteroid dehydrogenase 1 (HSD11B1);peroxisomal biogenesis factor 6 (PEX6); ras-related C3 botulinum toxinsubstrate 2 (RAC2); synovial sarcoma, X breakpoint 5 (SSX5); andacyl-Coenzyme A binding domain containing 3 (ACBD3); and instructionsfor use of the nucleic acid to detect the presence of the gene or theexpression level of the gene.

Another preferred embodiment of the invention provides a kit comprisingan antibody that binds to a protein produced by any one of the genesselected from the group consisting of erythroblastic leukemia viraloncogene homolog 2 (ERBB2); SMAD family member 4 (SMAD4); phosphataseand tensin homolog (PTEN); epidermal growth factor receptor oncogene(EGFR); histone-lysine N-methyltransferase (EZH2); SET domain containing2 (SETD2); von Hippel-Lindau tumor suppressor (VHL); pterin-4alpha-carbinolamine dehydratase/dimerization cofactor of hepatocytenuclear factor 1 alpha (PCBD1); protein phosphatase 2, regulatorysubunit B, gamma isoform (PPP2R2C); neural precursor cell expressed,developmentally downregulated 4 (NEDD4); prolyl 4-hydroxylase, alphapolypeptide II (P4HA2); SLC2A4 regulator (SLC2A4RG); sulfatase 2(SULF2); lysosomal protein transmembrane 4 alpha (LAPTM4A);3′-phosphoadenosine 5′-phosphosulfate synthase 2 (PAPSS2); aldo-ketoreductase family 1, member C1 (dihydrodiol dehydrogenase 1; 20-alpha(3-alpha)-hydroxysteroid dehydrogenase) (AKR1C1); protein tyrosinephosphatase, non-receptor type 12 (PTPN12); DCN1, defective in cullinneddylation 1, domain containing 4 (S. cerevisiae) (DCUN1D4);ras-related C3 botulinum toxin substrate 2 (RAC2); acyl-Coexzyme Adehydrogenase, C-4 to C-112 straight chain (ACADM); Rho GTPaseactivating protein 4 (ARHGAP4); ATPase type 13A1 (ATP13A1); chemokinereceptor 7 (CCR7); coronin 7 (CORO7); CXXC finger 4 (CXXC4);differentially expressed in FDCP 6 homolog (DEF6); KRI1 homolog (KRI1);limb region 1 homolog (LMBR1L); leukotriene B4 receptor (LTB4R);RAD54-like 2 (RAD54L2); chromosome X open reading frame 21 (CXorf21);SREBF chaperone (SCAP); selectin L (SELL); splicing factor 3a, subunit 2(SF3A2); Lyrm7 homolog (LYRM7); O-linked N-acetylglucosamine transferase(OGT); tubulin, alpha 3c (TUBA3C); tubulin, alpha 3d (TUBA3D); KH-typesplicing regulatory protein (KHSRP); DEAH (Asp-Glu-Ala-His) boxpolypeptide 30 (DHX30); APEX nuclease (apurinic/apyrimidinicendonuclease) 2 (APEX2); abhydrolase domain containing 14A (ABHD14A);UDP-glucose dehydrogenase (UGDH); H2A histone family, member Y2(H2AFY2); myosin VC (MYO5C); nephronectin (NPNT); KIAA1598 (KIAA1598);serglycin (SRGN); collagen, type VI, alpha 3 (COL6A3); G-proteinsignaling modulator 3 (GPSM3); hydroxysteroid dehydrogenase 1 (HSD11B1);peroxisomal biogenesis factor 6 (PEX6); ras-related C3 botulinum toxinsubstrate 2 (RAC2); synovial sarcoma, X breakpoint 5 (SSX5); andacyl-Coenzyme A binding domain containing 3 (ACBD3); and instructionsfor use of the antibody to detect the presence of the gene or theexpression level of the gene.

Another preferred embodiment of the invention provides a method forpredicting whether a breast cancer patient will respond to treatmentwith a histone deacetylase (HDAC) inhibitor comprising the steps of:

a) measuring the expression level of each of the following genes:transforming growth factor beta-3 (TGFB3); CD44 molecule (Indian bloodgroup) (CD44); cytochrome p450, family 4, subfamily Z, polypeptide 2pseudogene (CYP4Z2P); interferon-induced protein 44 (IFI44); solutecarrier family 9, subfamily A (NHE6, cation proton antiporter 6), member6 (SLC9A6); v-erb-b2 erythroblastic leukemia viral oncogene homolog 2,neuro/glioblastoma derived oncogene homolog (avian) (ERBB2); v-yes-1Yamaguchi sarcoma viral related oncogene homolog (LYN); pleckstrinhomology-like domain, family A, member 1 (PHLDA1); peroxisomeproliferator-activated receptor gamma (PPARG); dicarbonyl/L-xylulosereductase (DCXR); uridine phosphorylase 1 (UPP1); ATP-binding cassette,sub-family C (CFTR/MRP), member 11 (ABCC11); aldo-keto reductase family1, member C2 (dihydrodiol dehydrogenase 2; bile acid binding protein;3-alpha hydroxysteroid dehydrogenase, type III) (AKR1C2);BCL2-associated athanogene 2 (BAG2); TLR4 interactor with leucine-richrepeats (TRIL); uncharacterized LOC440335 (LOC440335); inhibin, beta B(INHBB); dickkopf 1 homolog (Xenopus laevis) (DKK1); insulin receptorsubstrate 2 (IRS2); chromosome 17 open reading frame 28 (C17orf28); LIMdomain kinase 2 (LIMK2); like-glycosyltransferase (LARGE); coiled-coildomain containing 82 (CCDC82); solute carrier family 40 (iron-regulatedtransporter), member 1 (SLC40A1); interferon-induced protein withtetratricopeptide repeats 1 (IFIT1); formin-like 2 (FMNL2); leukemiainhibitory factor (LIF); transforming growth factor, beta recetor 2(70/80 kDa) (TGFBR2); G protein-coupled receptor 160 (GPR160); cytokineinducible SH2-containing protein (CISH); phospholipase C, beta 4(PLCB4); B-cell linker (BLNK); phospholipase C, gamma 2(phosphatidylinositol-specific) (PLCG2); caveolin 2 (CAV2); prolinedehydrogenase (oxidase) 1 (PRODH); ras homolog family member B (RHOB);interferon-induced protein with tetratricopeptide repeats 3 (IFIT3);calbindin 2 (CALB2); TSPY-like 5 (TSPYL5); chromosome X open readingframe 61 (CXorf61); hematopoietically expressed homeobox (HHEX); cAMPresponsive element binding protein 3-like4 (CREB3L4); X-box bindingprotein 1 (XBP1); SAM pointed domain containing ets trsanscriptionfactor (SPDEF); nuclear receptor coactivator 7 (NCOA7); galaninprepropeptide (GAL); HECT and RLD domain containing E3 ubiquitin proteinligase 5 (HERC5); major histocompatibility complex, class I, A (HLA-A);centromere protein V (CENPV); frequently rearranged in advanced T-celllymphomas 2 (FRAT2); phospholipase B domain containing 1 (PLBD1);adenosine A2b receptor (ADORA2B); G protein-coupled receptor, family C,group 5, member A (GPRC5A); enoyl CoA hydratase domain containing 1(ECHDC1); guanylate binding protein 1, interferon-inducible (GBP1);sulfatase 2 (SULF2), uncharacterized LOC100507463 (LOC100507463), andKIAA1324 (KIAA1324) in a biological sample from the breast cancerpatient; and

b) correlating a high expression level, as compared to a normalized geneexpression level of the gene, of the following genes TGFB3, CYP4Z2P,ERBB2, DCXR, ABCC11, TRIL, LOC440335, INHBB, C17orf28, LIMK2, LARGE,SLC40A1, GPR160, CISH, PLCB4, BLNK, PRODH, RHOB, CREB3L4, XBP1, SPDEF,FRAT2, and KIAA1324 as an indication that the patient will respond tosuch treatment; and correlating a low expression level, as compared to anormalized gene expression level of the gene, of the following genesCD44, IFI44, SLC9A6, LYN, PHLDA1, PPARG, UPP1, AKR1C2, BAG2, DKK1, IRS2,IFIT1, FMNL2, LIF, TGFBR2, PLCG2, CAV2, IFIT3, CALB2, TSPYL5, CXorf61,HHEX, NCOA7, GAL, HERC5, HLA-A, CENPV, PLBD1, ADORA2B, GPRC5A, ECHDC1,GBP1, SULF2, and LOC100507463 as an indication that the patient willrespond to such treatment. In a further preferred embodiment of theinvention, the method further comprises the step of administering to thepatient a therapeutically effective amount of a HDAC inhibitor.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a picture of the 58 gene signature heatmap in 59 CCLE breastcancer cell lines.

FIGS. 2 (A and B) are pictures of heatmaps showing a correlation betweenCompound A breast cancer sensitivity biomarkers from 59 cell lines (A)to 352 breast tumors (B).

FIGS. 3 (A and B) are pictures of heatmaps showing a lack of acorrelation between Compound A breast cancer sensitivity biomarkers from59 cell lines (A) to 293 colon tumors (B).

FIGS. 4 (A, B, C, D, E, F, G, H, and I) shows graphs of clinical markersfor Compound A breast cancer sensitivity biomarkers. ER means estrogenreceptor; PR means progesterone receptor; HER2 means human epidermalgrowth factor receptor 2, which is also known as Neu, ErbB-2, CD340, orp185.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “a biomarker” means one biomarker or morethan one biomarker.

The terms “administer” or “administration” refer to the act of injectingor otherwise physically delivering a substance as it exists outside thebody (e.g., a formulation of the invention) into a patient, such as bymucosal, intradermal, intravenous, intramuscular delivery and/or anyother method of physical delivery described herein or known in the art.When a disease, or a symptom thereof, is being treated, administrationof the substance typically occurs after the onset of the disease orsymptoms thereof. When a disease, or symptoms thereof, are beingprevented, administration of the substance typically occurs before theonset of the disease or symptoms thereof.

Unless specifically indicated otherwise, the term “antibody,” as usedherein, shall be understood to encompass antibody molecules comprisingtwo immunoglobulin heavy chains and two immunoglobulin light chains(i.e., “full antibody molecules”) as well as antigen-binding fragmentsthereof. The terms “antigen-binding portion” of an antibody,“antigen-binding fragment” of an antibody, and the like, as used herein,include any naturally occurring, enzymatically obtainable, synthetic, orgenetically engineered polypeptide or glycoprotein that specificallybinds an antigen to form a complex. Antigen-binding fragments of anantibody may be derived, e.g., from full antibody molecules using anysuitable standard techniques such as proteolytic digestion orrecombinant genetic engineering techniques involving the manipulationand expression of DNA encoding antibody variable and (optionally)constant domains. Such DNA is known and/or is readily available from,e.g., commercial sources, DNA libraries (including, e.g., phage-antibodylibraries), or can be synthesized. The DNA may be sequenced andmanipulated chemically or by using molecular biology techniques, forexample, to arrange one or more variable and/or constant domains into asuitable configuration, or to introduce codons, create cysteineresidues, modify, add or delete amino acids, etc.

An antibody that “binds” an antigen of interest is one capable ofbinding that antigen with sufficient affinity such that the antibody isuseful in detecting the presence of the antigen.

The term “biological sample” shall generally mean any biological sampleobtained from an individual, body fluid, cell line, tissue culture, orother source. Body fluids are, for example, blood, lymph, sera, plasma,urine, semen, synovial fluid, and spinal fluid. Methods for obtainingtissue biopsies and body fluids from mammals are well known in the art.If the term “sample” is used alone, it shall still mean that the“sample” is a “biological sample”, i.e., the terms are usedinterchangeably.

The terms “composition” and “formulation” are intended to encompass aproduct containing the specified ingredients (e.g., an HDAC inhibitor)in, optionally, the specified amounts, as well as any product whichresults, directly or indirectly, from the combination of the specifiedingredients in, optionally, the specified amounts.

The term “excipients” refers to inert substances that are commonly usedas a diluent, vehicle, preservative, binder, stabilizing agent, etc. fordrugs and includes, but is not limited to, proteins (e.g., serumalbumin, etc), amino acids (e.g., aspartic acid, glutamic acid, lysine,arginine, glycine, histidine, etc.), fatty acids and phospholipids(e.g., alkyl sulfonates, caprylate, etc.), surfactants (e.g., SDS,polysorbate, nonionic surfactant, etc.), saccharides (e.g., sucrose,maltose, trehalose, etc.) and polyols (e.g., mannitol, sorbitol, etc.).See, also, Remington's Pharmaceutical Sciences (1990) Mack PublishingCo., Easton, Pa., which is hereby incorporated by reference in itsentirety.

The phrases “respond to treatment with a HDAC inhibitor” or “respond totreatment with a HDAC6 inhibitor” or similar phrases refer to theclinical benefit imparted to a patient suffering from a disease orcondition, such as cancer, from or as a result of the treatment with theHDAC inhibitor (e.g., a HDAC6 inhibitor). A clinical benefit includes acomplete remission, a partial remission, a stable disease (withoutprogression), progression-free survival, disease free survival,improvement in the time-to-progression (of the disease), improvement inthe time-to-death, or improvement in the overall survival time of thepatient from or as a result of the treatment with the HDAC inhibitor.There are criteria for determining a response to therapy and thosecriteria allow comparisons of the efficacy to alternative treatments(Slapak and Kufe, Principles of Cancer Therapy, in Harrisons'sPrinciples of Internal Medicine, 13th edition, eds. Isselbacher et al.,McGraw-Hill, Inc., 1994). For example, a complete response or completeremission of cancer is the disappearance of all detectable malignantdisease. A partial response or partial remission of cancer may be, forexample, an approximately 50 percent decrease in the product of thegreatest perpendicular diameters of one or more lesions or where thereis not an increase in the size of any lesion or the appearance of newlesions.

The term “progression of cancer” includes and may refer to metastasis, arecurrence of cancer, or an at least approximately 25 percent increasein the product of the greatest perpendicular diameter of one lesion orthe appearance of new lesions. The progression of cancer is “inhibited”if recurrence or metastasis of the cancer is reduced, slowed, delayed,or prevented.

“Nucleotides” are nucleosides that further include a phosphate groupcovalently linked to the sugar portion of the nucleoside. For those“nucleosides” that include a pentofuranosyl sugar, the phosphate groupcan be linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. A“nucleotide” is the “monomeric unit” of an “oligonucleotide” or a“polynucleotide”. Nucleotides are the units of deoxyribonucleic acid(DNA) and ribonucleic acid (RNA). As used herein, the term“polynucleotide” is synonymous with “nucleic acid”.

The term “probe” refers to synthetically or biologically producednucleic acids (DNA or RNA) which, by design or selection, containspecific nucleotide sequences that allow them to hybridize under definedpredetermined stringencies specifically (i.e., preferentially) to“nucleic acids”. A “probe” can be identified as a “capture probe”meaning that it “captures” the nucleic acid so that it can be separatedfrom undesirable materials which might obscure its detection. Onceseparation is accomplished, detection of the captured “target nucleicacid” can be achieved using a suitable procedure. “Capture probes” areoften already attached to a solid phase.

The term hybridization under “stringent conditions” is given the samemeaning as in Sambrook et al. (Molecular Cloning, A Laboratory Manual,Cold Spring Harbor Laboratory Press (1989), paragraph 1.101-1.104). Forexample, a “stringent hybridization” is the case when a hybridizationsignal is still detectable after washing for 1 hour with 1×SSC and 0.1%SDS at 50° C., preferably at 55° C., more preferably at 62° C., and mostpreferably at 68° C., and more preferably for 1 hour with 0.2×SSC and0.1% SDS at 50° C., preferably at 55° C., more preferably at 62° C., andmost preferably at 68° C. The composition of the SSC buffer is describedin Sambrook et al. (Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Laboratory Press (1989)).

A “transcribed polynucleotide” is a polynucleotide (e.g., a RNA, a cDNA,or an analog of one of an RNA or cDNA) that is complementary to orhomologous with all or a portion of a mature RNA made by transcriptionof a gene, such as the marker gene, and normal post-transcriptionalprocessing (e.g., splicing), if any, of the transcript. The term “cDNA”is an abbreviation for complementary DNA, the single-stranded ordouble-stranded DNA copy of a mRNA. The term “mRNA” is an abbreviationfor messenger RNA, the RNA that serves as a template for proteinsynthesis.

The terms “marker gene” or “biomarker gene” include a gene that isuseful for identifying a cancer patient that will or is likely torespond to treatment with a HDAC inhibitor, alone or in combination withanother cancer treatment.

The terms “marker polynucleotide” or “biomarker polynucleotide” includea nucleotide transcript (hnRNA or mRNA) encoded by a biomarker gene, orcDNA derived from the nucleotide transcript, or a segment of saidtranscript or cDNA.

The terms “marker protein”, “marker polypeptide”, “biomarker protein”,or “biomarker polypeptide” include a protein or polypeptide encoded by abiomarker gene or a fragment thereof.

The terms “marker” and “biomarker” are used interchangeably and refer toa biomarker gene, biomarker polynucleotide, or biomarker protein, asdefined above.

The term “gene product” refers to a biomarker polynucleotide, orbiomarker protein encoded by a biomarker gene.

The expression of a biomarker gene differs from the level of expressionof the biomarker gene in a reference sample or a normalized geneexpression level if the level of expression of the biomarker gene in asample from the patient differs from the level in a sample from thereference subject or a normalized gene expression level by an amountgreater than the standard error of the assay employed to assessexpression, and preferably at least 10%, and more preferably 25%, 50%,75%, 100%, 125%, 150%, 175%, 200%, 300%, 400%, 500% or 1,000% of thatamount. For example, expression of the biomarker gene in the patient canbe considered “lower” than the level of expression in a control subjector a normalized gene expression level if the level of expression in asample from the patient is lower than the level in a sample from thecontrol subject or a normalized gene expression level by an amountgreater than the standard error of the assay employed to assessexpression, and preferably at least 10%, and more preferably 25%, 50%,75%, 100%, 125%, 150%, 175%, 200%, 300%, 400%, 500% or 1,000% of thatamount. Alternatively, expression of the biomarker gene in the patientcan be considered “higher” than the level of expression in a controlsubject or a normalized gene expression level if the level of expressionin a sample from the patient is higher than the level in a sample fromthe control subject or a normalized gene expression level by an amountgreater than the standard error of the assay employed to assessexpression, and preferably at least 10%, and more preferably 25%, 50%,75%, 100%, 125%, 150%, 175%, 200%, 300%, 400%, 500% or 1,000% of thatamount.

The terms “level of expression” or “expression level” are usedinterchangeably and generally refer to the amount of a polynucleotide oran amino acid product or protein in a biological sample. “Expression”generally refers to the process by which gene encoded information isconverted into the structures present and operating in the cell.Therefore, the “expression” of a gene may refer to transcription into apolynucleotide, translation into a protein or even posttranslationalmodification of the protein. Fragments of the transcribedpolynucleotide, the translated protein or the postranslationallymodified protein shall also be regarded as expressed whether theyoriginate from a transcript generated by alternative splicing, adegraded transcript, or from a posttranslational processing of theprotein, e.g., by proteolysis. “Expressed genes” include those that aretranscribed into a polynucleotide as mRNA and then translated into aprotein; and also include expressed genes that are transcribed into RNAbut not translated into a protein (for example, transfer and ribosomalRNAs).

The term “mutation” refers to a change or alteration in a gene orprotein, such as an insertion, deletion, substitution, or modificationof one or more nucleic acids or amino acids in the gene or protein,respectively. For example, the mutation may be a single point mutation,multiple point mutations, a frame-shift mutation, deletion, insertion,inversion, or DNA expression mutation.

The terms “overexpression”, “increased expression” or “high expression”refer to an upward deviation in levels of expression, as compared to thebaseline expression level in a sample used as a control or a normalizedgene expression level or normalized protein expression level.

The terms “underexpression”, “decreased expression” or “low expression”refer to a downward deviation in levels of expression, as compared tothe baseline expression level in a sample used as a control or anormalized gene expression level or normalized protein expression level.

The phrase “normalized gene expression level” refers to an average geneexpression level between individual cell lines.

The phrase “normalized protein expression level” refers to an averageprotein expression level between individual cell lines.

A “kit” is any manufacture (e.g., a package or container) comprising atleast one reagent, e.g., a probe, for specifically detecting a biomarkergene or protein. The manufacture is preferably promoted, distributed, orsold as a unit for performing the methods of the invention.

The term “therapy” refers to any protocol, method, and/or agent that canbe used in the prevention, management, treatment, and/or amelioration ofa disease.

The term “alkyl,” as used herein, refers to saturated, straight- orbranched-chain hydrocarbon moieties containing, in certain embodiments,between one and six, or one and eight carbon atoms, respectively.Examples of C₁-C₆ alkyl moieties include, but are not limited to,methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, neopentyl,n-hexyl moieties; and examples of C₁-C₈ alkyl moieties include, but arenot limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl,neopentyl, n-hexyl, heptyl, and octyl moieties.

The term “alkenyl,” as used herein, denotes a monovalent group derivedfrom a hydrocarbon moiety containing, in certain embodiments, from twoto six, or two to eight carbon atoms having at least one carbon-carbondouble bond. The double bond may or may not be the point of attachmentto another group. Alkenyl groups include, but are not limited to, forexample, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, heptenyl,octenyl and the like.

The term “alkynyl,” as used herein, denotes a monovalent group derivedfrom a hydrocarbon moiety containing, in certain embodiments, from twoto six, or two to eight carbon atoms having at least one carbon-carbontriple bond. The alkynyl group may or may not be the point of attachmentto another group. Representative alkynyl groups include, but are notlimited to, for example, ethynyl, 1-propynyl, 1-butynyl, heptynyl,octynyl and the like.

The term “alkoxy” refers to an —O-alkyl moiety.

The term “aryl,” as used herein, refers to a mono- or poly-cycliccarbocyclic ring system having one or more aromatic rings, fused ornon-fused, including, but not limited to, phenyl, naphthyl,tetrahydronaphthyl, indanyl, idenyl and the like.

The term “aralkyl,” or “arylalkyl,” as used herein, refers to an alkylresidue attached to an aryl ring. Examples include, but are not limitedto, benzyl, phenethyl and the like.

The term “carbocyclic,” as used herein, denotes a monovalent groupderived from a monocyclic or polycyclic saturated, partiallyunsaturated, or fully unsaturated carbocyclic ring compound. Examples ofcarbocyclic groups include groups found in the cycloalkyl definition andaryl definition.

The term “cycloalkyl,” as used herein, denotes a monovalent groupderived from a monocyclic or polycyclic saturated or partially unsaturedcarbocyclic ring compound. Examples of C₃-C₈-cycloalkyl include, but notlimited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cyclopentyl and cyclooctyl; and examples of C₃-C₁₂-cycloalkyl include,but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,bicyclo[2.2.1]heptyl, and bicyclo[2.2.2]octyl. Also contemplated aremonovalent groups derived from a monocyclic or polycyclic carbocyclicring compound having at least one carbon-carbon double bond by theremoval of a single hydrogen atom. Examples of such groups include, butare not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl,cyclohexenyl, cycloheptenyl, cyclooctenyl, and the like.

The term “heteroaryl,” as used herein, refers to a mono- or poly-cyclic(e.g., bi-, or tri-cyclic or more) fused or non-fused, moieties or ringsystem having at least one aromatic ring, having from five to ten ringatoms of which one ring atom is selected from S, O and N; zero, one ortwo ring atoms are additional heteroatoms independently selected from S,O and N; and the remaining ring atoms are carbon. Heteroaryl includes,but is not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl,pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl,oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl,benzimidazolyl, benzooxazolyl, quinoxalinyl, and the like.

The term “heteroaralkyl,” as used herein, refers to an alkyl residueattached to a heteroaryl ring. Examples include, but are not limited to,pyridinylmethyl, pyrimidinylethyl and the like.

The term “heterocycloalkyl,” as used herein, refers to a non-aromatic3-, 4-, 5-, 6- or 7-membered ring or a bi- or tri-cyclic group fused ofnon-fused system, where (i) each ring contains between one and threeheteroatoms independently selected from oxygen, sulfur and nitrogen,(ii) each 5-membered ring has 0 to 1 double bonds and each 6-memberedring has 0 to 2 double bonds, (iii) the nitrogen and sulfur heteroatomsmay optionally be oxidized, (iv) the nitrogen heteroatom may optionallybe quaternized, and (iv) any of the above rings may be fused to abenzene ring. Representative heterocycloalkyl groups include, but arenot limited to, [1,3]dioxolane, pyrrolidinyl, pyrazolinyl,pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl,oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl,isothiazolidinyl, and tetrahydrofuryl.

The term “alkylamino” refers to a group having the structure —NH(C₁-C₁₂alkyl) where C₁-C₁₂ alkyl is as previously defined.

The term “acyl” includes residues derived from acids, including but notlimited to carboxylic acids, carbamic acids, carbonic acids, sulfonicacids, and phosphorous acids. Examples include aliphatic carbonyls,aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphaticsulfinyls, aromatic phosphates and aliphatic phosphates. Examples ofaliphatic carbonyls include, but are not limited to, acetyl, propionyl,2-fluoroacetyl, butyryl, 2-hydroxy acetyl, and the like.

In accordance with the invention, any of the aryls, substituted aryls,heteroaryls and substituted heteroaryls described herein, can be anyaromatic group. Aromatic groups can be substituted or unsubstituted.

The terms “hal,” “halo” and “halogen,” as used herein, refer to an atomselected from fluorine, chlorine, bromine and iodine.

The term “oxo” as used herein, refers to an oxygen that is attached to acarbon, preferably by a double bond (e.g., carbonyl).

As described herein, compounds used in the methods of the invention mayoptionally be substituted with one or more substituents, such as areillustrated generally in formula I, or as exemplified by particularclasses, subclasses, and species of the invention. It will beappreciated that the phrase “optionally substituted” is usedinterchangeably with the phrase “substituted or unsubstituted”. Ingeneral, the term “substituted”, whether preceded by the term“optionally” or not, refers to the replacement of hydrogen radicals in agiven structure with the radical of a specified substituent. Unlessotherwise indicated, an optionally substituted group may have asubstituent at each substitutable position of the group, and when morethan one position in any given structure may be substituted with morethan one substituent selected from a specified group, the substituentmay be either the same or different at every position. The terms“optionally substituted”, “optionally substituted alkyl,” “optionallysubstituted “optionally substituted alkenyl,” “optionally substitutedalkynyl”, “optionally substituted cycloalkyl,” “optionally substitutedcycloalkenyl,” “optionally substituted aryl”, “optionally substitutedheteroaryl,” “optionally substituted aralkyl”, “optionally substitutedheteroaralkyl,” “optionally substituted heterocycloalkyl,” and any otheroptionally substituted group as used herein, refer to groups that aresubstituted or unsubstituted by independent replacement of one, two, orthree or more of the hydrogen atoms thereon with substituents including,but not limited to:

alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycloalkyl, heteroaryl,arylalkyl, hetero arylalkyl,

—F, —Cl, —Br, —I,

—OH, protected hydroxy, oxygen, oxo,

—NO₂, —CN,

—NH₂, protected amino, —NH—C₁-C₁₂-alkyl, —NH-aryl, -dialkylamino,

—O—C₁-C₁₂-alkyl, —O-aryl,

—C(O)—, —C(O)O—, —C(O)NH—, —OC(O)—, —OC(O)O—, —OC(O)NH—, —NHC(O)—,—NHC(O)O—,

—C(O)—C₁-C₁₂-alkyl, —C(O)—C₃-C₁₂-cycloalkyl, —C(O)-aryl,—C(O)-heteroaryl, —C(O)-heterocycloalkyl,

—C(O)O—C₁-C₁₂-alkyl, —C(O)O—C₃-C₁₂-cycloalkyl, —C(O)O-aryl,—C(O)O-heteroaryl, —C(O)O-heterocycloalkyl,

—CONH₂, —CONH—C₁-C₁₂-alkyl, —CONH-aryl,

—OCO₂—C₁-C₁₂-alkyl, —OCO₂-aryl, —OCONH₂, —OCONH—C₁-C₁₂-alkyl,—OCONH-aryl,

—NHC(O)—C₁-C₁₂-alkyl, —NHC(O)-aryl, —NHCO₂—C₁-C₁₂-alkyl, —NHCO₂— aryl,

—S(O)—C₁-C₁₂-alkyl, —S(O)-aryl, —SO₂NH—C₁-C₁₂-alkyl, —SO₂NH— aryl,

—NHSO₂—C₁-C₁₂-alkyl, —NHSO₂-aryl,

—SH, —S—C₁-C₁₂-alkyl, or —S-aryl.

In certain embodiments, the optionally substituted groups include thefollowing: C₁-C₁₂-alkyl, C₂-C₁₂-alkenyl, C₂-C₁₂-alkynyl,C₃-C₁₂-cycloalkyl, C₃-C₁₂-aryl, C₃-C₁₂-heterocycloalkyl,C₃-C₁₂-heteroaryl, C₄-C₁₂-arylalkyl, or C₂-C₁₂-heteroarylalkyl.

It is understood that the aryls, heteroaryls, alkyls, and the like canbe further substituted.

As used herein, the term “metal chelator” refers to any molecule ormoiety that is capable of forming a complex (i.e., “chelates”) with ametal ion. In certain exemplary embodiments, a metal chelator refers toany molecule or moiety that “binds” to a metal ion, in solution, makingit unavailable for use in chemical/enzymatic reactions. In certainembodiments, the solution comprises aqueous environments underphysiological conditions. Examples of metal ions include, but are notlimited to, Ca²⁺, Fe³⁺, Zn²⁺, Na⁺, etc. In certain embodiments, themetal chelator binds Zn²⁺. In certain embodiments, molecules of moietiesthat precipitate metal ions are not considered to be metal chelators.

As used herein, the term “small molecule” refers to a non-peptidic,non-oligomeric organic compound either synthesized in the laboratory orfound in nature. Small molecules, as used herein, can refer to compoundsthat are “natural product-like”, however, the term “small molecule” isnot limited to “natural product-like” compounds. Rather, a smallmolecule is typically characterized in that it contains severalcarbon-carbon bonds, and has a molecular weight of less than 1500,although this characterization is not intended to be limiting for thepurposes of the present invention. Examples of “small molecules” thatoccur in nature include, but are not limited to, taxol, dynemicin, andrapamycin. In certain other preferred embodiments, natural-product-likesmall molecules are utilized.

The terms “subject” or “patient” as used herein refer to a mammal. Asubject therefore refers to, for example, dogs, cats, horses, cows,pigs, guinea pigs, and the like. Preferably the subject is a human.

“Treat”, “treating” and “treatment” refer to a method of alleviating orabating a disease and/or its attendant symptoms.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts of the compounds formed by the process disclosed hereinwhich are, within the scope of sound medical judgment, suitable for usein contact with the tissues of humans and lower animals without unduetoxicity, irritation, allergic response and the like, and arecommensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, S. M. Berge, etal. describes pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 66: 1-19 (1977).

As used herein, the term “pharmaceutically acceptable ester” refers toesters of the compounds formed by the process disclosed herein whichhydrolyze in vivo and include those that break down readily in the humanbody to leave the parent compound or a salt thereof. Suitable estergroups include, for example, those derived from pharmaceuticallyacceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic,cycloalkanoic and alkanedioic acids, in which each alkyl or alkenylmoiety advantageously has not more than 6 carbon atoms. Examples ofparticular esters include, but are not limited to, formates, acetates,propionates, butyrates, acrylates and ethylsuccinates.

The term “pharmaceutically acceptable prodrugs” as used herein refers tothose prodrugs of the compounds formed by the process disclosed hereinwhich are, within the scope of sound medical judgment, suitable for usein contact with the tissues of humans and lower animals with unduetoxicity, irritation, allergic response, and the like, commensurate witha reasonable benefit/risk ratio, and effective for their intended use,as well as the zwitterionic forms, where possible, of the compounds ofthe present invention. “Prodrug”, as used herein means a compound whichis convertible in vivo by metabolic means (e.g. by hydrolysis) to affordany compound delineated by the formulae disclosed herein. Various formsof prodrugs are known in the art, for example, as discussed inBundgaard, (ed.), Design of Prodrugs, Elsevier (1985); Widder, et al.(ed.), Methods in Enzymology, vol. 4, Academic Press (1985);Krogsgaard-Larsen, et al., (ed). “Design and Application of Prodrugs,Textbook of Drug Design and Development, Chapter 5, 113-191 (1991);Bundgaard, et al., Journal of Drug Deliver Reviews, 8:1-38 (1992);Bundgaard, J. of Pharmaceutical Sciences, 77:285 et seq. (1988); Higuchiand Stella (eds.) Prodrugs as Novel Drug Delivery Systems, AmericanChemical Society (1975); and Bernard Testa & Joachim Mayer, “HydrolysisIn Drug And Prodrug Metabolism Chemistry, Biochemistry And Enzymology,”John Wiley and Sons, Ltd. (2002).

Combinations of substituents and variables envisioned by this inventionare only those that result in the formation of stable compounds. Theterm “stable”, as used herein, refers to compounds which possessstability sufficient to allow manufacture and which maintains theintegrity of the compound for a sufficient period of time to be usefulfor the purposes detailed herein (e.g., therapeutic or prophylacticadministration to a subject).

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is substantially or essentially free from components thatnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. Particularly, in embodiments thecompound is at least 85% pure, more preferably at least 90% pure, morepreferably at least 95% pure, and most preferably at least 99% pure.

Methods

The invention provides methods for using a biomarker to identify acancer patient who will or is likely to respond to treatment.Specifically, the invention relates to the use of one or more of threeassociation studies of cancer types, gene mutations, or gene expressionlevels in order to identify a cancer patient who will or is likely torespond to treatment with a histone deacetylase (HDAC) inhibitor, aloneor in combination with another cancer treatment. The methods may,optionally, further include treating such patient with an HDACinhibitor, alone or in combination with another cancer treatment.

An embodiment of the invention comprises an association study of cancertypes, which associates treatment effect by tumor type.

Another embodiment of the invention comprises an association study ofgene mutations, which associates gene mutation analysis with treatmenttype and tumor type.

A further embodiment of the invention comprises an association study ofgene expression levels, which associates gene expression analysis withtumor type.

An embodiment of the invention provides a method for using a biomarkerto identify a cancer patient who will or is likely to respond totreatment with a histone deacetylase (HDAC) inhibitor, alone or incombination with another cancer treatment, by interpreting data from oneor more of the three association studies of cancer types, genemutations, or gene expression levels.

An embodiment of the invention provides a method for using a biomarkerto predict whether a cancer patient will or is likely to respond totreatment with a histone deacetylase (HDAC) inhibitor, alone or incombination with another cancer treatment, by interpreting data from oneor more of the three association studies of cancer types, genemutations, or gene expression levels.

An embodiment of the invention provides a method for using a biomarkerto predict the response of a cancer patient to treatment with a histonedeacetylase (HDAC) inhibitor, alone or in combination with anothercancer treatment, by interpreting data from one or more of the threeassociation studies of cancer types, gene mutations, or gene expressionlevels.

An embodiment of the invention provides a method for using a biomarkerto predict cancer cell inhibition in response to treatment with ahistone deacetylase (HDAC) inhibitor, alone or in combination withanother cancer treatment, by interpreting data from one or more of thethree association studies of cancer types, gene mutations, or geneexpression levels.

An embodiment of the invention provides a method for using a biomarkerto predict tumor cell killing in response to treatment with a histonedeacetylase (HDAC) inhibitor, alone or in combination with anothercancer treatment, by interpreting data from one or more of the threeassociation studies of cancer types, gene mutations, or gene expressionlevels.

An embodiment of the invention provides a method for using a biomarkerto predict the sensitivity of cancer cell growth to treatment with ahistone deacetylase (HDAC) inhibitor, alone or in combination withanother cancer treatment, by interpreting data from one or more of thethree association studies of cancer types, gene mutations, or geneexpression levels.

An embodiment of the invention provides a method for using a biomarkerto predict the sensitivity of tumor cell growth to treatment with ahistone deacetylase (HDAC) inhibitor, alone or in combination withanother cancer treatment, by interpreting data from one or more of thethree association studies of cancer types, gene mutations, or geneexpression levels.

An embodiment of the invention provides a method for using a biomarkerto identify a cancer patient who is likely to benefit from treatmentwith a histone deacetylase (HDAC) inhibitor, alone or in combinationwith another cancer treatment, by interpreting data from one or more ofthe three association studies of cancer types, gene mutations, or geneexpression levels.

An embodiment of the invention provides a method for treating cancercomprising administering a histone deacetylase (HDAC) inhibitor, aloneor in combination with another cancer treatment, to a patient who,according to data from one or more of the three association studies ofcancer types, gene mutations, or gene expression levels, will or islikely to respond to such treatment.

An embodiment of the invention provides a method for treating cancer ina patient comprising the steps of predicting whether a cancer patientwill respond to treatment with a histone deacetylase (HDAC) inhibitor,alone or in combination with another cancer treatment, by interpretingdata from one or more of the three association studies of cancer types,gene mutations, or gene expression levels, and administering to thepatient a therapeutically effective amount of a histone deacetylase(HDAC) inhibitor, alone or in combination with another cancer treatment,if it is determined that the patient will or is likely to respond tosuch treatment.

An embodiment of the invention provides a method for re-treating cancerin a patient comprising the steps of predicting whether a cancer patientwill respond to treatment with a histone deacetylase (HDAC) inhibitor,alone or in combination with another cancer treatment, by interpretingdata from one or more of the three association studies of cancer types,gene mutations, or gene expression levels, and administering to thepatient a therapeutically effective amount of a histone deacetylase(HDAC) inhibitor, alone or in combination with another cancer treatment,if it is determined that the patient will or is likely to respond tosuch treatment.

An embodiment of the invention provides a method for modifying cancertreatment in a patient comprising the steps of predicting whether acancer patient will respond to treatment with a histone deacetylase(HDAC) inhibitor, alone or in combination with another cancer treatment,by interpreting data from one or more of the three association studiesof cancer types, gene mutations, or gene expression levels, andadministering to the patient a therapeutically effective amount of ahistone deacetylase (HDAC) inhibitor, alone or in combination withanother cancer treatment, if it is determined that the patient will oris likely to respond to such treatment.

An embodiment of the invention provides a method for optimizing cancertreatment in a patient comprising the steps of predicting whether acancer patient will respond to treatment with a histone deacetylase(HDAC) inhibitor, alone or in combination with another cancer treatment,by interpreting data from one or more of the three association studiesof cancer types, gene mutations, or gene expression levels, andadministering to the patient a therapeutically effective amount of ahistone deacetylase (HDAC) inhibitor, alone or in combination withanother cancer treatment, if it is determined that the patient will oris likely to respond to such treatment.

An embodiment of the invention provides a method for treating breastcancer in a patient in need thereof comprising the steps of:

a) determining whether the human epidermal growth factor receptor 2(Her2) protein is overexpressed, as compared to a normalized proteinexpression level of the protein, in a biological sample from the breastcancer patient;

b) correlating the presence of such overexpression as an indication thatthe patient will respond to such treatment, or correlating the absenceof such overexpression as an indication that the patient will notrespond to such treatment; and

c) administering to a patient that will respond to treatment atherapeutically effective amount of a histone deacetylase (HDAC)inhibitor.

An embodiment of the invention provides a method for treating cancer ina patient in need thereof comprising the steps of:

a) measuring the expression level of a gene selected from the groupconsisting of pterin-4 alpha-carbinolamine dehydratase/dimerizationcofactor of hepatocyte nuclear factor 1 alpha (PCBD1); proteinphosphatase 2, regulatory subunit B, gamma isoform (PPP2R2C); neuralprecursor cell expressed, developmentally downregulated 4 (NEDD4);prolyl 4-hydroxylase, alpha polypeptide II (P4HA2); SLC2A4 regulator(SLC2A4RG); sulfatase 2 (SULF2); lysosomal protein transmembrane 4 alpha(LAPTM4A); 3′-phosphoadenosine 5′-phosphosulfate synthase 2 (PAPSS2);aldo-keto reductase family 1, member C1 (dihydrodiol dehydrogenase 1;20-alpha (3-alpha)-hydroxysteroid dehydrogenase) (AKR1C1); proteintyrosine phosphatase, non-receptor type 12 (PTPN12); DCN1, defective incullin neddylation 1, domain containing 4 (S. cerevisiae) (DCUN1D4);ras-related C3 botulinum toxin substrate 2 (RAC2); acyl-Coexzyme Adehydrogenase, C-4 to C-112 straight chain (ACADM); Rho GTPaseactivating protein 4 (ARHGAP4); ATPase type 13A1 (ATP13A1); chemokinereceptor 7 (CCR7); coronin 7 (CORO7); CXXC finger 4 (CXXC4);differentially expressed in FDCP 6 homolog (DEF6); KRI1 homolog (KRI1);limb region 1 homolog (LMBR1L); leukotriene B4 receptor (LTB4R);RAD54-like 2 (RAD54L2); chromosome X open reading frame 21 (CXorf21);SREBF chaperone (SCAP); selectin L (SELL); splicing factor 3a, subunit 2(SF3A2); Lyrm7 homolog (LYRM7); O-linked N-acetylglucosamine transferase(OGT); tubulin, alpha 3c (TUBA3C); tubulin, alpha 3d (TUBA3D); KH-typesplicing regulatory protein (KHSRP); DEAH (Asp-Glu-Ala-His) boxpolypeptide 30 (DHX30); APEX nuclease (apurinic/apyrimidinicendonuclease) 2 (APEX2); and abhydrolase domain containing 14A (ABHD14A)in a biological sample from the cancer patient;

b) correlating a low expression level, as compared to a normalized geneexpression level of the gene, of any one or more of the genes PCBD1,PPP2R2C, NEDD4, P4HA2, SLC2A4RG, SULF2, LAPTM4A, PAPSS2, AKR1C1, PTPN12,and DCUN1D4 as an indication that the patient will respond to suchtreatment; or correlating a high expression level, as compared to anormalized gene expression level of the gene, of any one or more of thegenes RAC2, ACADM, ARHGAP4, ATP13A1, CCR7, CORO7, CXXC4, DEF6, KRI1,LMBR1L, LTB4R, RAD54L2, CXorf21, SCAP, SELL, SF3A2, LYRM7, OGT, TUBA3C,TUBA3D, KHSRP, DHX30, APEX2, and ABHD14A as an indication that thepatient will respond to such treatment; and

c) administering to a patient that will respond to treatment atherapeutically effective amount of a histone deacetylase (HDAC)inhibitor.

An embodiment of the invention provides a method for treating colorectalcancer in a patient in need thereof comprising the steps of:

a) determining whether a gene mutation in the SMAD family member 4(SMAD4) gene is present in a biological sample from the colorectalcancer patient;

b) correlating the presence of a gene mutation as an indication that thepatient will respond to such treatment, or correlating the absence of agene mutation as an indication that the patient will not respond to suchtreatment; and

c) administering to a patient that will respond to treatment atherapeutically effective amount of a histone deacetylase (HDAC)inhibitor and a proteasome inhibitor.

An embodiment of the invention provides a method for treating cancer ina patient in need thereof comprising the steps of:

a) determining whether a gene mutation in a gene selected from the groupconsisting of phosphatase and tensin homolog (PTEN), epidermal growthfactor receptor oncogene (EGFR), histone-lysine N-methyltransferase(EZH2), SET domain containing 2 (SETD2), and von Hippel-Lindau tumorsuppressor (VHL), is present in a biological sample from the cancerpatient;

b) correlating the presence of one or more such mutations as anindication that the patient will respond to such treatment, orcorrelating the absence of such mutations as an indication that thepatient will not respond to such treatment; and

c) administering to a patient that will respond to treatment atherapeutically effective amount of a histone deacetylase (HDAC)inhibitor and a proteasome inhibitor.

An embodiment of the invention provides a method for treating cancer ina patient in need thereof comprising the steps of:

a) measuring the expression level of a gene selected from the groupconsisting of UDP-glucose dehydrogenase (UGDH); H2A histone family,member Y2 (H2AFY2); myosin VC (MYO5C); nephronectin (NPNT); KIAA1598(KIAA1598); serglycin (SRGN); collagen, type VI, alpha 3 (COL6A3);G-protein signaling modulator 3 (GPSM3); hydroxysteroid dehydrogenase 1(HSD11B1); peroxisomal biogenesis factor 6 (PEX6); ras-related C3botulinum toxin substrate 2 (RAC2); synovial sarcoma, X breakpoint 5(SSX5); and acyl-Coenzyme A binding domain containing 3 (ACBD3); in abiological sample from the cancer patient;

b) correlating a low expression level, as compared to a normalized geneexpression level of the gene, of any one or more of the genes UGDH,H2AFY2, MYO5C, NPNT, and KIAA1598 as an indication that the patient willrespond to such treatment; or correlating a high expression level of thegene, as compared to a normalized gene expression level, of any one ormore of the genes SRGN, COL6A3, GPSM3, HSD11B1, PEX6, RAC2, SSX5, andACBD3 as an indication that the patient will respond to such treatment;and

c) administering to a patient that will respond to treatment atherapeutically effective amount of a histone deacetylase (HDAC)inhibitor and a proteasome inhibitor.

An embodiment of the invention provides a method for treating cancer ina patient in neee thereof comprising the steps of:

a) evaluating the data from one or more association studies comprising:

-   -   1) an association study that associates treatment effect by        tumor type, wherein brain/neuron cancer, breast cancer, lymphoid        cancer, kidney cancer, colon/large intestine cancer, and skin        cancer are determined to be sensitive to combination treatment        with a histone deacetylase inhibitor and a proteasome inhibitor,    -   2) an association study that associates gene mutation analysis        with treatment type and tumor type, wherein        -   i) the overexpression of the human epidermal growth factor            receptor 2 (Her2) protein, as compared to a normalized            protein expression level of the protein, in a biological            sample from a breast cancer patient is an indication that            the patient will respond to treatment with a histone            deacetylase inhibitor,        -   ii) the presence of a gene mutation in the SMAD family            member 4 (SMAD4) gene in a biological sample from a            colorectal cancer patient is an indication that the patient            will respond to combination treatment with a histone            deacetylase inhibitor and a proteasome inhibitor, or        -   iii) the presence of one or more gene mutations in a gene            selected from the group consisting of phosphatase and tensin            homolog (PTEN), epidermal growth factor receptor oncogene            (EGFR), histone-lysine N-methyltransferase (EZH2), SET            domain containing 2 (SETD2), and von Hippel-Lindau tumor            suppressor (VHL) in a biological sample from a cancer            patient is an indication that the patient will respond to            combination treatment with a histone deacetylase inhibitor            and a proteasome inhibitor, or    -   3) an association study that associates gene expression analysis        with treatment type, comprising        -   i) measuring the expression level of a gene selected from            the group consisting of pterin-4 alpha-carbinolamine            dehydratase/dimerization cofactor of hepatocyte nuclear            factor 1 alpha (PCBD1); protein phosphatase 2, regulatory            subunit B, gamma isoform (PPP2R2C); neural precursor cell            expressed, developmentally downregulated 4 (NEDD4); prolyl            4-hydroxylase, alpha polypeptide II (P4HA2); SLC2A4            regulator (SLC2A4RG); sulfatase 2 (SULF2); lysosomal protein            transmembrane 4 alpha (LAPTM4A); 3′-phosphoadenosine            5′-phosphosulfate synthase 2 (PAPSS2); aldo-keto reductase            family 1, member C1 (dihydrodiol dehydrogenase 1; 20-alpha            (3-alpha)-hydroxysteroid dehydrogenase) (AKR1C1); protein            tyrosine phosphatase, non-receptor type 12 (PTPN12); DCN1,            defective in cullin neddylation 1, domain containing 4 (S.            cerevisiae) (DCUN1D4); ras-related C3 botulinum toxin            substrate 2 (RAC2); acyl-Coexzyme A dehydrogenase, C-4 to            C-112 straight chain (ACADM); Rho GTPase activating protein            4 (ARHGAP4); ATPase type 13A1 (ATP13A1); chemokine receptor            7 (CCR7); coronin 7 (CORO7); CXXC finger 4 (CXXC4);            differentially expressed in FDCP 6 homolog (DEF6); KRI1            homolog (KRI1); limb region 1 homolog (LMBR1L); leukotriene            B4 receptor (LTB4R); RAD54-like 2 (RAD54L2); chromosome X            open reading frame 21 (CXorf21); SREBF chaperone (SCAP);            selectin L (SELL); splicing factor 3a, subunit 2 (SF3A2);            Lyrm7 homolog (LYRM7); O-linked N-acetylglucosamine            transferase (OGT); tubulin, alpha 3c (TUBA3C); tubulin,            alpha 3d (TUBA3D); KH-type splicing regulatory protein            (KHSRP); DEAH (Asp-Glu-Ala-His) box polypeptide 30 (DHX30);            APEX nuclease (apurinic/apyrimidinic endonuclease) 2            (APEX2); and abhydrolase domain containing 14A (ABHD14A) in            a biological sample from the cancer patient; and            -   a) correlating a low expression level, as compared to a                normalized gene expression level of the gene, of any one                or more of the genes PCBD1, PPP2R2C, NEDD4, P4HA2,                SLC2A4RG, SULF2, LAPTM4A, PAPSS2, AKR1C1, PTPN12, and                DCUN1D4 as an indication that the patient will respond                to treatment with a histone deacetylase inhibitor; or                correlating a high expression level, as compared to a                normalized gene expression level of the gene, of any one                or more of the genes RAC2, ACADM, ARHGAP4, ATP13A1,                CCR7, CORO7, CXXC4, DEF6, KRI1, LMBR1L, LTB4R, RAD54L2,                CXorf21, SCAP, SELL, SF3A2, LYRM7, OGT, TUBA3C, TUBA3D,                KHSRP, DHX30, APEX2, and ABHD14A as an indication that                the patient will respond to treatment with a histone                deacetylase inhibitor, or        -   ii) measuring the expression level of a gene selected from            the group consisting of UDP-glucose dehydrogenase (UGDH);            H2A histone family, member Y2 (H2AFY2); myosin VC (MYO5C);            nephronectin (NPNT); KIAA1598 (KIAA1598); serglycin (SRGN);            collagen, type VI, alpha 3 (COL6A3); G-protein signaling            modulator 3 (GPSM3); hydroxysteroid dehydrogenase 1            (HSD11B1); peroxisomal biogenesis factor 6 (PEX6);            ras-related C3 botulinum toxin substrate 2 (RAC2); synovial            sarcoma, X breakpoint 5 (SSX5); and acyl-Coenzyme A binding            domain containing 3 (ACBD3); in a biological sample from the            cancer patient; and            -   a) correlating a low expression level, as compared to a                normalized gene expression level of the gene, of any one                or more of the genes UGDH, H2AFY2, MYO5C, NPNT, and                KIAA1598 as an indication that the patient will respond                to combination treatment with a histone deacetylase                inhibitor and a proteasome inhibitor; or correlating a                high expression level of the gene, as compared to a                normalized gene expression level, of any one or more of                the genes SRGN, COL6A3, GPSM3, HSD11B1, PEX6, RAC2,                SSX5, and ACBD3 as an indication that the patient will                respond to combination treatment with a histone                deacetylase inhibitor and a proteasome inhibitor,

b) evaluating the data regarding cancer types, gene mutations, or geneexpression levels to determine whether the patient will respond to suchtreatment; and

c) administering to a patient that will respond to treatment atherapeutically effective amount of a histone deacetylase (HDAC)inhibitor and a proteasome inhibitor.

An embodiment of the invention provides a method for treating breastcancer in a patient in need thereof comprising the steps of:

a) measuring the expression level of each of the following genes:transforming growth factor beta-3 (TGFB3); CD44 molecule (Indian bloodgroup) (CD44); cytochrome p450, family 4, subfamily Z, polypeptide 2pseudogene (CYP4Z2P); interferon-induced protein 44 (IFI44); solutecarrier family 9, subfamily A (NHE6, cation proton antiporter 6), member6 (SLC9A6); v-erb-b2 erythroblastic leukemia viral oncogene homolog 2,neuro/glioblastoma derived oncogene homolog (avian) (ERBB2); v-yes-1Yamaguchi sarcoma viral related oncogene homolog (LYN); pleckstrinhomology-like domain, family A, member 1 (PHLDA1); peroxisomeproliferator-activated receptor gamma (PPARG); dicarbonyl/L-xylulosereductase (DCXR); uridine phosphorylase 1 (UPP1); ATP-binding cassette,sub-family C (CFTR/MRP), member 11 (ABCC11); aldo-keto reductase family1, member C2 (dihydrodiol dehydrogenase 2; bile acid binding protein;3-alpha hydroxysteroid dehydrogenase, type III) (AKR1C2);BCL2-associated athanogene 2 (BAG2); TLR4 interactor with leucine-richrepeats (TRIL); uncharacterized LOC440335 (LOC440335); inhibin, beta B(INHBB); dickkopf 1 homolog (Xenopus laevis) (DKK1); insulin receptorsubstrate 2 (IRS2); chromosome 17 open reading frame 28 (C17orf28); LIMdomain kinase 2 (LIMK2); like-glycosyltransferase (LARGE); coiled-coildomain containing 82 (CCDC82); solute carrier family 40 (iron-regulatedtransporter), member 1 (SLC40A1); interferon-induced protein withtetratricopeptide repeats 1 (IFIT1); formin-like 2 (FMNL2); leukemiainhibitory factor (LIF); transforming growth factor, beta recetor 2(70/80 kDa) (TGFBR2); G protein-coupled receptor 160 (GPR160); cytokineinducible SH2-containing protein (CISH); phospholipase C, beta 4(PLCB4); B-cell linker (BLNK); phospholipase C, gamma 2(phosphatidylinositol-specific) (PLCG2); caveolin 2 (CAV2); prolinedehydrogenase (oxidase) 1 (PRODH); ras homolog family member B (RHOB);interferon-induced protein with tetratricopeptide repeats 3 (IFIT3);calbindin 2 (CALB2); TSPY-like 5 (TSPYL5); chromosome X open readingframe 61 (CXorf61); hematopoietically expressed homeobox (HHEX); cAMPresponsive element binding protein 3-like4 (CREB3L4); X-box bindingprotein 1 (XBP1); SAM pointed domain containing ets trsanscriptionfactor (SPDEF); nuclear receptor coactivator 7 (NCOA7); galaninprepropeptide (GAL); HECT and RLD domain containing E3 ubiquitin proteinligase 5 (HERC5); major histocompatibility complex, class I, A (HLA-A);centromere protein V (CENPV); frequently rearranged in advanced T-celllymphomas 2 (FRAT2); phospholipase B domain containing 1 (PLBD1);adenosine A2b receptor (ADORA2B); G protein-coupled receptor, family C,group 5, member A (GPRC5A); enoyl CoA hydratase domain containing 1(ECHDC1); guanylate binding protein 1, interferon-inducible (GBP1);sulfatase 2 (SULF2), uncharacterized LOC100507463 (LOC100507463), andKIAA1324 (KIAA1324) in a biological sample from the breast cancerpatient;

b) correlating a high expression level, as compared to a normalized geneexpression level of the gene, of the following genes TGFB3, CYP4Z2P,ERBB2, DCXR, ABCC11, TRIL, LOC440335, INHBB, C17orf28, LIMK2, LARGE,SLC40A1, GPR160, CISH, PLCB4, BLNK, PRODH, RHOB, CREB3L4, XBP1, SPDEF,FRAT2, and KIAA1324 as an indication that the patient will respond tosuch treatment; and correlating a low expression level, as compared to anormalized gene expression level of the gene, of the following genesCD44, IFI44, SLC9A6, LYN, PHLDA1, PPARG, UPP1, AKR1C2, BAG2, DKK1, IRS2,IFIT1, FMNL2, LIF, TGFBR2, PLCG2, CAV2, IFIT3, CALB2, TSPYL5, CXorf61,HHEX, NCOA7, GAL, HERC5, HLA-A, CENPV, PLBD1, ADORA2B, GPRC5A, ECHDC1,GBP1, SULF2, and LOC100507463 as an indication that the patient willrespond to such treatment;

c) administering to a patient that will respond to treatment atherapeutically effective amount of a histone deacetylase (HDAC)inhibitor.

An embodiment of the invention provides a histone deacetylase (HDAC)inhibitor for use in treating breast cancer in a patient whooverexpresses the human epidermal growth factor receptor 2 (Her2)protein, as compared to a normalized protein expression level of theprotein.

An embodiment of the invention provides the combination of a histonedeacetylase (HDAC) inhibitor and a proteasome inhibitor for use intreating colorectal cancer in a patient having a gene mutation in theSMAD family member 4 (SMAD4) gene.

An embodiment of the invention provides the combination of a histonedeacetylase (HDAC) inhibitor and a proteasome inhibitor for use intreating cancer in a patient having a gene mutation in a gene selectedfrom the group consisting of phosphatase and tensin homolog (PTEN),epidermal growth factor receptor oncogene (EGFR), histone-lysineN-methyltransferase (EZH2), SET domain containing 2 (SETD2), and vonHippel-Lindau tumor suppressor (VHL).

An embodiment of the invention provides a histone deacetylase (HDAC)inhibitor for use in treating cancer in a patient who has a lowexpression level, as compared to a normalized gene expression level ofthe gene, of any one or more of the genes PCBD1, PPP2R2C, NEDD4, P4HA2,SLC2A4RG, SULF2, LAPTM4A, PAPSS2, AKR1C1, PTPN12, and DCUN1D4, or a highexpression level, as compared to a normalized gene expression level ofthe gene, of any one or more of the genes RAC2, ACADM, ARHGAP4, ATP13A1,CCR7, CORO7, CXXC4, DEF6, KRI1, LMBR1L, LTB4R, RAD54L2, CXorf21, SCAP,SELL, SF3A2, LYRM7, OGT, TUBA3C, TUBA3D, KHSRP, DHX30, APEX2, andABHD14A.

An embodiment of the invention provides the combination of a histonedeacetylase (HDAC) inhibitor and a proteasome inhibitor for use intreating cancer in a patient who has a low expression level, as comparedto a normalized gene expression level of the gene, of any one or more ofthe genes UGDH, H2AFY2, MYO5C, NPNT, and KIAA1598, or a high expressionlevel, as compared to a normalized gene expression level, of any one ormore of the genes SRGN, COL6A3, GPSM3, HSD11B1, PEX6, RAC2, SSX5, andACBD3.

An embodiment of the invention provides a histone deacetylase (HDAC)inhibitor for use in treating breast cancer in a patient whooverexpresses the human epidermal growth factor receptor 2 (Her2)protein, as compared to a normalized protein expression level of theprotein, wherein the patient has been tested and found to have the Her2protein.

An embodiment of the invention provides the combination of a histonedeacetylase (HDAC) inhibitor and a proteasome inhibitor for use intreating colorectal cancer in a patient having a gene mutation in theSMAD family member 4 (SMAD4) gene, wherein the patient has been testedand found to have a mutation in the SMAD4 gene.

An embodiment of the invention provides the combination of a histonedeacetylase (HDAC) inhibitor and a proteasome inhibitor for use intreating cancer in a patient having a gene mutation in a gene selectedfrom the group consisting of phosphatase and tensin homolog (PTEN),epidermal growth factor receptor oncogene (EGFR), histone-lysineN-methyltransferase (EZH2), SET domain containing 2 (SETD2), and vonHippel-Lindau tumor suppressor (VHL), wherein the patient has beentested and found to have a mutation in any one of the PTEN, EGFR, EZH2,SETD2, and VHL genes.

An embodiment of the invention provides a histone deacetylase (HDAC)inhibitor for use in treating cancer in a patient who has a lowexpression level, as compared to a normalized gene expression level ofthe gene, of any one or more of the genes PCBD1, PPP2R2C, NEDD4, P4HA2,SLC2A4RG, SULF2, LAPTM4A, PAPSS2, AKR1C1, PTPN12, and DCUN1D4, or a highexpression level, as compared to a normalized gene expression level ofthe gene, of any one or more of the genes RAC2, ACADM, ARHGAP4, ATP13A1,CCR7, CORO7, CXXC4, DEF6, KRI1, LMBR1L, LTB4R, RAD54L2, CXorf21, SCAP,SELL, SF3A2, LYRM7, OGT, TUBA3C, TUBA3D, KHSRP, DHX30, APEX2, andABHD14A, wherein the patient has been tested and found to have a lowexpression level of any one or more of the genes PCBD1, PPP2R2C, NEDD4,P4HA2, SLC2A4RG, SULF2, LAPTM4A, PAPSS2, AKR1C1, PTPN12, and DCUN1D4, ora high expression level of any one or more of the genes RAC2, ACADM,ARHGAP4, ATP13A1, CCR7, CORO7, CXXC4, DEF6, KRI1, LMBR1L, LTB4R,RAD54L2, CXorf21, SCAP, SELL, SF3A2, LYRM7, OGT, TUBA3C, TUBA3D, KHSRP,DHX30, APEX2, and ABHD14A.

An embodiment of the invention provides the combination of a histonedeacetylase (HDAC) inhibitor and a proteasome inhibitor for use intreating cancer in a patient who has a low expression level, as comparedto a normalized gene expression level of the gene, of any one or more ofthe genes UGDH, H2AFY2, MYO5C, NPNT, and KIAA1598, or a high expressionlevel, as compared to a normalized gene expression level, of any one ormore of the genes SRGN, COL6A3, GPSM3, HSD11B1, PEX6, RAC2, SSX5, andACBD3, wherein the patient has been tested and found to have a lowexpression level of any one or more of the genes UGDH, H2AFY2, MYO5C,NPNT, and KIAA1598, or a high expression level of any one or more of thegenes UGDH, H2AFY2, MYO5C, NPNT, and KIAA1598.

An embodiment of the invention provides a histone deacetylase (HDAC)inhibitor for use in treating breast cancer in a patient wherein thetreatment comprises testing the patient for overexpression of the humanepidermal growth factor receptor 2 (Her2) protein, as compared to anormalized protein expression level of the protein, and administeringthe HDAC inhibitor if the patient overexpresses the Her2 protein.

An embodiment of the invention provides the combination of a histonedeacetylase (HDAC) inhibitor and a proteasome inhibitor for use intreating colorectal cancer in a patient wherein the treatment comprisestesting the patient for a gene mutation in the SMAD family member 4(SMAD4) gene and administering the combination if a mutation in theSMAD4 gene is found.

An embodiment of the invention provides the combination of a histonedeacetylase (HDAC) inhibitor and a proteasome inhibitor for use intreating cancer in a patient wherein the treatment comprises testing thepatient for a gene mutation in a gene selected from the group consistingof phosphatase and tensin homolog (PTEN), epidermal growth factorreceptor oncogene (EGFR), histone-lysine N-methyltransferase (EZH2), SETdomain containing 2 (SETD2), and von Hippel-Lindau tumor suppressor(VHL), and administering the combination if a mutation in any one of thePTEN, EGFR, EZH2, SETD2, and VHL genes is found.

An embodiment of the invention provides a histone deacetylase (HDAC)inhibitor for use in treating cancer in a patient wherein the treatmentcomprises testing the patient for a low expression level, as compared toa normalized gene expression level of the gene, of any one or more ofthe genes PCBD1, PPP2R2C, NEDD4, P4HA2, SLC2A4RG, SULF2, LAPTM4A,PAPSS2, AKR1C1, PTPN12, and DCUN1D4, or a high expression level, ascompared to a normalized gene expression level of the gene, of any oneor more of the genes RAC2, ACADM, ARHGAP4, ATP13A1, CCR7, CORO7, CXXC4,DEF6, KRI1, LMBR1L, LTB4R, RAD54L2, CXorf21, SCAP, SELL, SF3A2, LYRM7,OGT, TUBA3C, TUBA3D, KHSRP, DHX30, APEX2, and ABHD14A, and administeringthe combination if a low expression level of any one or more of thegenes PCBD1, PPP2R2C, NEDD4, P4HA2, SLC2A4RG, SULF2, LAPTM4A, PAPSS2,AKR1C1, PTPN12, and DCUN1D4, or a high expression level of any one ormore of the genes RAC2, ACADM, ARHGAP4, ATP13A1, CCR7, CORO7, CXXC4,DEF6, KRI1, LMBR1L, LTB4R, RAD54L2, CXorf21, SCAP, SELL, SF3A2, LYRM7,OGT, TUBA3C, TUBA3D, KHSRP, DHX30, APEX2, and ABHD14A is found.

An embodiment of the invention provides the combination of a histonedeacetylase (HDAC) inhibitor and a proteasome inhibitor for use intreating cancer in a patient wherein the treatment comprises testing thepatient for a low expression level, as compared to a normalized geneexpression level of the gene, of any one or more of the genes UGDH,H2AFY2, MYO5C, NPNT, and KIAA1598, or a high expression level, ascompared to a normalized gene expression level, of any one or more ofthe genes SRGN, COL6A3, GPSM3, HSD11B1, PEX6, RAC2, SSX5, and ACBD3, andadministering the combination if a low expression level of any one ormore of the genes UGDH, H2AFY2, MYO5C, NPNT, and KIAA1598, or a highexpression level of any one or more of the genes UGDH, H2AFY2, MYO5C,NPNT, and KIAA1598 is found.

An embodiment of the invention provides a method of testing forresponsiveness of a patient to treatment with a histone deacetylase(HDAC) inhibitor, the method comprising testing for overexpression, ascompared to a normalized protein expression level of the protein, of thehuman epidermal growth factor receptor 2 (Her2) protein. In a specificembodiment, the testing is performed in vitro.

An embodiment of the invention provides for use of the overexpression,as compared to a normalized protein expression level of the protein, ofthe human epidermal growth factor receptor 2 (Her2) protein forassessing responsiveness of a patient to treatment with a histonedeacetylase (HDAC) inhibitor.

An embodiment of the invention provides for use of a probe for the humanepidermal growth factor receptor 2 (Her2) protein for assessingresponsiveness of a patient to treatment with a histone deacetylase(HDAC) inhibitor.

An embodiment of the invention provides the overexpression, as comparedto a normalized protein expression level of the protein, of the humanepidermal growth factor receptor 2 (Her2) protein for use in treatingbreast cancer in a patient in need thereof wherein the treatmentcomprises identifying the overexpression of the Her2 protein in apatient and then administering a histone deacetylase (HDAC) inhibitor ifthe patient is found to overexpress the Her2 protein.

An embodiment of the invention provides a gene mutation in the SMADfamily member 4 (SMAD4) gene for use in treating colorectal cancer in apatient in need thereof wherein the treatment comprises identifying amutation in the SMAD4 gene in a patient and then administering thecombination of a histone deacetylase (HDAC) inhibitor and a proteasomeinhibitor if the patient is found to have a mutation in the SMAD4 gene.

An embodiment of the invention provides a gene mutation in a geneselected from the group consisting of phosphatase and tensin homolog(PTEN), epidermal growth factor receptor oncogene (EGFR), histone-lysineN-methyltransferase (EZH2), SET domain containing 2 (SETD2), and vonHippel-Lindau tumor suppressor (VHL) for use in treating cancer in apatient in need thereof wherein the treatment comprises identifying amutation in any one of the PTEN, EGFR, EZH2, SETD2, and VHL genes in apatient wherein the treatment comprises identifying the presence of themutation in any one of the PTEN, EGFR, EZH2, SETD2, and VHL genes andthen administering a histone deacetylase (HDAC) inhibitor and aproteasome inhibitor if the patient is found to have a mutation in anyone of the PTEN, EGFR, EZH2, SETD2, and VHL genes.

An embodiment of the invention provides a probe for the human epidermalgrowth factor receptor 2 (Her2) protein for use in treating breastcancer in a patient in need thereof wherein the treatment comprisesusing the probe to identify the overexpression, as compared to anormalized protein expression level of the protein, of the Her2 proteinin a patient and then administering a histone deacetylase (HDAC)inhibitor if the patient is found to overexpress the Her2 protein.

In certain embodiments of the methods and uses described above, theHDAC6 inhibitor is a compound of formula I:

or a pharmaceutically acceptable salt, ester or prodrug thereof.

In certain embodiments of the methods and uses described above, theHDAC6 inhibitor is Compound A.

Association Study of Cancer Types

An embodiment of the invention comprises an association study of cancertypes, which associates treatment effect by tumor type.

Preferably, numerous cancer cell lines are used in the associationstudy. For example, the cell lines can be specific for breast cancer,hematologic cancer, colorectal cancer, lung cancer, skin cancer, braincancer, renal cancer, liver cancer, prostate cancer, ovarian cancer, andstomach cancer. However, cell lines specific for other forms of cancermay be used. In addition, multiple cell lines that are specific for oneform of cancer, such as breast cancer, may be used.

In this association study of cancer types, each cell line is culturedand treated with an HDAC inhibitor, with or without another cancertreatment. The viabilities of the cell lines are then measured at theend of the treatments. Viability may be measured by any means known inthe art. Preferably, the sensitivities of the cell lines are describedusing the concentration of HDAC inhibitor that provides 50% inhibitionof cell viabilities (IC₅₀).

For example, a specific embodiment of this study that is described inmore detail in the Examples determined that brain/neuronal cancer,breast cancer, lymphoid cancer, kidney cancer, colon cancer, cancer ofthe large intestine, and skin cancer were each sensitive to combinationtreatment with a HDAC inhibitor and another cancer treatment. Inaddition, the specific embodiment of this study determined thathematologic malignant cancer was the most sensitive to treatment withboth a HDAC inhibitor alone, or combination treatment with a HDACinhibitor and another cancer treatment.

Association Study of Gene Mutations

Another embodiment of the invention comprises an association study ofgene mutations, which associates gene mutation analysis with treatmenttype and tumor type.

Preferably, numerous cancer cell lines are used in the associationstudy. For example, the cell lines can be specific for breast cancer,hematologic cancer, colorectal cancer, lung cancer, skin cancer, braincancer, renal cancer, liver cancer, prostate cancer, ovarian cancer, andstomach cancer. However, cell lines specific for other forms of cancermay be used. In addition, multiple cell lines that are specific for oneform of cancer, such as breast cancer, may be used.

In this association study of gene mutations, each cell line is culturedand treated with an HDAC inhibitor, with or without another cancertreatment. The viabilities of the cell lines are then measured at theend of the treatments. Viability may be measured by any means known inthe art. Preferably, the sensitivities of the cell lines are describedusing the concentration of HDAC inhibitor that provides 50% inhibitionof cell viabilities (IC₅₀).

Preferably numerous oncogene/tumor suppressor genes are analyzed togenerate the results of this study. For example, in the present methods,numerous common oncogene/tumor supporessor genes can be analyzed.

The genes are analyzed to determine an association between the genotypeand the cell sensitivities to the treatments.

The mutation profiles of the cell lines may be analyzed by any meansknown in the art. Preferably, the mutation profiles of the cell linesare downloaded from the Sanger COSMIC DB on the Internet: www dot sangerdot ac dot uk/cosmic.

The statistical significance of the association between the genotype andthe cell sensitivities to the treatments may be analyzed by any meansknown in the art. For each gene locus, it is preferable to use the“Fisher's Exact Test” to calculate statistical significance (p<0.05) ofassociation between the genotype and the cell sensitivities to thetreatments.

For example, a specific embodiment of this study that is described inmore detail in the Examples correlated epidermal growth factor receptor2 (Her2) protein overexpression, as compared to a normalized proteinexpression level of the protein, caused by erythroblastic leukemia viraloncogene homolog 2 (ERBB2) gene amplification or by any other means, ina biological sample from a breast cancer patient as an indication thatthe patient will respond to treatment with an HDAC inhibitor. Thespecific embodiment of this study also correlated the presence of a genemutation in the SMAD family member 4 (SMAD4) gene in a biological samplefrom a colorectal cancer patient as an indication that the patient willrespond to combination treatment with a HDAC inhibitor and anothercancer treatment. The specific embodiment of this study furthercorrelated the presence of a gene mutation in a gene selected from thegroup consisting of phosphatase and tensin homolog (PTEN), epidermalgrowth factor receptor oncogene (EGFR), histone-lysineN-methyltransferase (EZH2), SET domain containing 2 (SETD2), and vonHippel-Lindau tumor suppressor (VHL) in a biological sample from acancer patient as an indication that the patient will respond tocombination treatment with a HDAC inhibitor and another cancertreatment.

However, the specific embodiment of this study correlated the presenceof the basal-type or triple negative mutations (estrogenreceptor-negative, progesterone receptor-negative and HER2/neu-negative)in a biological sample from a breast cancer patient as an indicationthat the patient will not respond to treatment with an HDAC inhibitor.

Association Study of Gene Expression

A further embodiment of the invention comprises an association study ofgene expression levels, which associates gene expression levels withtumor type.

Preferably, numerous cancer cell lines are used in the associationstudy. For example, the cell lines can be specific for breast cancer,hematologic cancer, colorectal cancer, lung cancer, skin cancer, braincancer, renal cancer, liver cancer, prostate cancer, ovarian cancer, andstomach cancer. However, cell lines specific for other forms of cancermay be used. In addition, multiple cell lines that are specific for oneform of cancer, such as breast cancer, may be used.

In this association study of gene expression, each cell line is culturedand treated with an HDAC inhibitor, with or without another cancertreatment. The viabilities of the cell lines are then measured at theend of the treatments. Viability may be measured by any means known inthe art. Preferably, the sensitivities of the cell lines are describedusing the concentration of HDAC inhibitor that provides 50% inhibitionof cell viabilities (IC₅₀).

The gene expression profiles of the cell lines used in the study shouldbe obtained. These may be obtained by any means known in the art.Preferably, the gene expression profiles are obtained from theArrayExpress (AE) database on the Internet: www dot ebi dot ac dotuk/arrayexpress/, which is maintained by the European BioinformaticsInstitute. Alternatively, the gene expression profiles are obtained fromthe caArray database on the Internet: <URL:https://cabig.nci.nihgov/caArray_GSKdata/>, which is from NIH. More preferably, the geneexpression profiles of the cell lines are obtained from a combination ofthe above two databases.

Next, the expression intensity of the individual genes should beobtained and normalized between individual cell lines. This may be doneby any means known in the art. It is preferable to download theAffymetrix CEL files, which contain the expression intensity ofindividual genes from these public databases, and then use theR/BioConductor packages on the Internet: <URL:http://bioconductor.org>in order to normalize the gene-expression values between individual celllines. The correlation coefficient and its significance (p<0.001) may becalculated using the CORTEST function of R package between the IC₅₀values and the normalized gene expression levels for each probe-set. Forvalidation, only genes with all probe sets showing significantcorrelations should be reported; and genes should be removed if any ofits probe-sets are non-significant.

Alternatively, the level of expression of a biomarker gene in a samplemay be assessed by detecting the level of expression of a biomarkerprotein encoded by the biomarker gene. The level of expression of thebiomarker protein may be detected using a reagent that specificallybinds with the marker protein. Preferably, the reagent is selected fromthe group consisting of an antibody, a fragment of an antibody, and anantibody derivative.

There are many different types of immunoassays that may be used in themethod of the present invention, e.g., enzyme linked immunoabsorbentassay (ELISA), fluorescent immunosorbent assay (FIA), chemical linkedimmunosorbent assay (CLIA), radioimmuno assay (RIA), and immunoblotting.For a review of the different immunoassays which may be used, see:Lottspeich and Zorbas (eds.), Bioanalytik, 1^(st) edition 1998, SpektrumAkademischer Verlag, Heidelberg, Berlin, Germany. Therefore, the levelof expression may be determined using a method selected from the groupconsisting of proteomics, flow cytometry, immunocytochemistry,immunohistochemistry, enzyme-linked immunosorbent assay, multi-channelenzyme-linked immunosorbent assay, and variations of these methods.

Alternatively, the level of expression of a biomarker gene in abiological sample may be assessed by detecting the level of expressionof a transcribed biomarker polynucleotide encoded by the biomarker gene.For example, the transcribed biomarker polynucleotide is a cDNA, mRNA orhnRNA.

The step of detecting may further comprise amplifying the transcribedpolynucleotide. The amplification can be performed with the polymerasechain reaction which specifically amplifies nucleic acids to detectableamounts. Other possible amplification reactions are the Ligase ChainReaction (LCR; Wu D. Y. and Wallace R. B., Genomics 4 (1989) 560-569;and Barany F., Proc. Natl. Acad. Sci. USA 88 (1991)189-193); PolymeraseLigase Chain Reaction (Barany F., PCR Methods and Applic. 1 (1991)5-16); Gap-LCR (WO 90/01069); Repair Chain Reaction (EP 0439182 A2), 3SR(Kwoh, D. Y. et al., Proc. Natl. Acad. Sci. USA 86 (1989) 1173-1177;Guatelli, J. C. et al., Proc. Natl. Acad. Sci. USA 87 (1990) 1874-1878;WO 92/08808), and NASBA (U.S. Pat. No. 5,130,238). Further, there arestrand displacement amplification (SDA), transcription mediatedamplification (TMA), and QP-amplification (for a review see e.g. Whelen,A. C. and Persing, D. H., Annu. Rev. Microbiol. 50 (1996) 349-373;Abramson, R. D. and Myers T. W., Curr. Opin. Biotechnol. 4 (1993)41-47). For example, the step of detecting can use the method ofquantitative reverse transcriptase polymerase chain reaction.

Other suitable polynucleotide detection methods are known in the fieldand are described in standard textbooks, such as Sambrook J. et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989; and Ausubel, F. et al., CurrentProtocols in Molecular Biology, 1987, J. Wiley and Sons, NY. There maybe also further purification steps before the polynucleotide detectionstep is carried out as, for example, a precipitation step. The detectionmethods may include but are not limited to the binding or intercalatingof specific dyes as ethidiumbromide, which intercalates into thedouble-stranded polynucleotides and changes their fluorescencethereafter. The purified polynucleotide may also be separated byelectrophoretic methods optionally after a restriction digest andvisualized thereafter. There are also probe-based assays which exploitthe oligonucleotide hybridisation to specific sequences and subsequentdetection of the hybrid. It is also possible to sequence the DNA afterfurther steps known in the field. A preferred template-dependent DNApolymerase is Taq polymerase.

Alternatively, the level of expression of a biomarker gene is assessedby detecting the presence of the transcribed marker polynucleotide in asample with a probe that anneals with the transcribed markerpolynucleotide under stringent hybridization conditions. This method maybe performed in a homogeneous assay system. An example of a“homogeneous” assay system is the TaqMan™ system that has been detailedin U.S. Pat. No. 5,210,015, U.S. Pat. No. 5,804,375 and U.S. Pat. No.5,487,972. Briefly, the method is based on a double-labelled probe andthe 5′-3′ exonuclease activity of Taq DNA polymerase. The probe iscomplementary to the target sequence to be amplified by the PCR processand is located between the two PCR primers during each polymerisationcycle step. The probe has two fluorescent labels attached to it. One isa reporter dye, such as 6-carboxyfluorescein (FAM), which has itsemission spectra quenched by energy transfer due to the spatialproximity of a second fluorescent dye, 6-carboxy-tetramethyl-rhodamine(TAMRA). In the course of each amplification cycle, the Taq DNApolymerase in the process of elongating a primed DNA strand displacesand degrades the annealed probe, the latter due to the intrinsic 5′-3′exonuclease activity of the polymerase. The mechanism also frees thereporter dye from the quenching activity of TAMRA. As a consequence, thefluorescent activity increases with an increase in cleavage of theprobe, which is proportional to the amount of PCR product formed.Accordingly, an amplified target sequence is measured by detecting theintensity of released fluorescence label. Another example for“homogeneous” assay systems are provided by the formats used in theLightCycler™ instrument (see e.g. U.S. Pat. No. 6,174,670), some of themsometimes called “kissing probe” formats. Again, the principle is basedon two interacting dyes which, however, are characterized in that theemission wavelength of a donor-dye excites an acceptor-dye byfluorescence resonance energy transfer. The COBAS™ AmpliPrep instrument(Roche Diagnostics GmbH, D-68305 Mannheim, Germany) was introduced toexpand automation by isolating target sequences using biotinylatedsequence-specific capture probes along with streptavidin-coated magneticparticles (Jungkind, D., J. Clin. Virol. 20 (2001) 1-6; Stelzl, E. etal., J. Clin. Microbiol. 40 (2002) 1447-1450). It has lately been joinedby an additional versatile tool, the Total Nucleic Acid Isolation (TNAI)Kit (Roche Diagnostics). This laboratory-use reagent allows the generic,not sequence-specific isolation of all nucleic acids from plasma andserum on the COBAS™ AmpliPrep instrument based essentially on the methoddeveloped by Boom, R. et al., J. Clin. Microbiol. 28 (1990) 495-503.

For example, a specific embodiment of this study that is described inmore detail in the Examples correlated a low expression level, ascompared to a normalized gene expression level of the gene, of any oneor more of the genes selected from the group consisting of pterin-4alpha-carbinolamine dehydratase/dimerization cofactor of hepatocytenuclear factor 1 alpha (PCBD1); protein phosphatase 2, regulatorysubunit B, gamma isoform (PPP2R2C); neural precursor cell expressed,developmentally downregulated 4 (NEDD4); prolyl 4-hydroxylase, alphapolypeptide II (P4HA2); SLC2A4 regulator (SLC2A4RG); sulfatase 2(SULF2); lysosomal protein transmembrane 4 alpha (LAPTM4A);3′-phosphoadenosine 5′-phosphosulfate synthase 2 (PAPSS2); aldo-ketoreductase family 1, member C1 (dihydrodiol dehydrogenase 1; 20-alpha(3-alpha)-hydroxysteroid dehydrogenase) (AKR1C1); protein tyrosinephosphatase, non-receptor type 12 (PTPN12); and DCN1, defective incullin neddylation 1, domain containing 4 (S. cerevisiae) (DCUN1D4) in abiological sample from a cancer patient as an indication that thepatient will respond to treatment with an HDAC inhibitor. The specificembodiment of this study also correlated a high expression level, ascompared to a normalized gene expression level of the gene, of any oneor more of the genes selected from the group consisting of ras-relatedC3 botulinum toxin substrate 2 (RAC2); acyl-Coexzyme A dehydrogenase,C-4 to C-112 straight chain (ACADM); Rho GTPase activating protein 4(ARHGAP4); ATPase type 13A1 (ATP13A1); chemokine receptor 7 (CCR7);coronin 7 (CORO7); CXXC finger 4 (CXXC4); differentially expressed inFDCP 6 homolog (DEF6); KRI1 homolog (KRI1); limb region 1 homolog(LMBR1L); leukotriene B4 receptor (LTB4R); RAD54-like 2 (RAD54L2);chromosome X open reading frame 21 (CXorf21); SREBF chaperone (SCAP);selectin L (SELL); splicing factor 3a, subunit 2 (SF3A2); Lyrm7 homolog(LYRM7); O-linked N-acetylglucosamine transferase (OGT); tubulin, alpha3c (TUBA3C); tubulin, alpha 3d (TUBA3D); KH-type splicing regulatoryprotein (KHSRP); DEAH (Asp-Glu-Ala-His) box polypeptide 30 (DHX30); APEXnuclease (apurinic/apyrimidinic endonuclease) 2 (APEX2); and abhydrolasedomain containing 14A (ABHD14A) in a biological sample from a cancerpatient as an indication that the patient will respond to treatment withan HDAC inhibitor. In addition, the specific embodiment of this studycorrelated a low expression level, as compared to a normalized geneexpression level of the gene, of any one or more of the genes selectedfrom the group consisting of UDP-glucose dehydrogenase (UGDH); H2Ahistone family, member Y2 (H2AFY2); myosin VC (MYO5C); nephronectin(NPNT); and KIAA1598 (KIAA1598) in a biological sample from a cancerpatient as an indication that the patient will respond to combinationtreatment with an HDAC inhibitor and another cancer treatment. Further,the specific embodiment of this study correlated a high expressionlevel, as compared to a normalized gene expression level of the gene, ofany one or more of the genes selected from the group consisting ofserglycin (SRGN); collagen, type VI, alpha 3 (COL6A3); G-proteinsignaling modulator 3 (GPSM3); hydroxysteroid dehydrogenase 1 (HSD11B1);peroxisomal biogenesis factor 6 (PEX6); ras-related C3 botulinum toxinsubstrate 2 (RAC2); synovial sarcoma, X breakpoint 5 (SSX5); andacyl-Coenzyme A binding domain containing 3 (ACBD3) in a biologicalsample from a cancer patient as an indication that the patient willrespond to combination treatment with an HDAC inhibitor and anothercancer treatment.

58 Gene Signature Prediction Model for Breast Cancer

An embodiment of the invention comprises a 58 gene signature predictionmodel that can be used to identify breast cancer patients that willrespond to treatment with an HDAC inhibitor.

Breast cancer cell lines were studied to produce a 58 gene signatureprediction model using a correlation analysis of their IC50 values andtheir gene expression data published at the CCLE database (www dot broadinstitute dot org/ccle/home).

For the 58 genes in the “signature”, 35 low expression and 23 highexpression genes related to the “sensitive” signature, while 35 highexpression and 23 low expression related to the “resistant” signature(see FIG. 1). The names of the 58 genes are: transforming growth factorbeta-3 (TGFB3); CD44 molecule (Indian blood group) (CD44); cytochromep450, family 4, subfamily Z, polypeptide 2 pseudogene (CYP4Z2P);interferon-induced protein 44 (IFI44); solute carrier family 9,subfamily A (NHE6, cation proton antiporter 6), member 6 (SLC9A6);v-erb-b2 erythroblastic leukemia viral oncogene homolog 2,neuro/glioblastoma derived oncogene homolog (avian) (ERBB2); v-yes-1Yamaguchi sarcoma viral related oncogene homolog (LYN); pleckstrinhomology-like domain, family A, member 1 (PHLDA1); peroxisomeproliferator-activated receptor gamma (PPARG); dicarbonyl/L-xylulosereductase (DCXR); uridine phosphorylase 1 (UPP1); ATP-binding cassette,sub-family C (CFTR/MRP), member 11 (ABCC11); aldo-keto reductase family1, member C2 (dihydrodiol dehydrogenase 2; bile acid binding protein;3-alpha hydroxysteroid dehydrogenase, type III) (AKR1C2);BCL2-associated athanogene 2 (BAG2); TLR4 interactor with leucine-richrepeats (TRIL); uncharacterized LOC440335 (LOC440335); inhibin, beta B(INHBB); dickkopf 1 homolog (Xenopus laevis) (DKK1); insulin receptorsubstrate 2 (IRS2); chromosome 17 open reading frame 28 (C17orf28); LIMdomain kinase 2 (LIMK2); like-glycosyltransferase (LARGE); coiled-coildomain containing 82 (CCDC82); solute carrier family 40 (iron-regulatedtransporter), member 1 (SLC40A1); interferon-induced protein withtetratricopeptide repeats 1 (IFIT1); formin-like 2 (FMNL2); leukemiainhibitory factor (LIF); transforming growth factor, beta recetor 2(70/80 kDa) (TGFBR2); G protein-coupled receptor 160 (GPR160); cytokineinducible SH2-containing protein (CISH); phospholipase C, beta 4(PLCB4); B-cell linker (BLNK); phospholipase C, gamma 2(phosphatidylinositol-specific) (PLCG2); caveolin 2 (CAV2); prolinedehydrogenase (oxidase) 1 (PRODH); ras homolog family member B (RHOB);interferon-induced protein with tetratricopeptide repeats 3 (IFIT3);calbindin 2 (CALB2); TSPY-like 5 (TSPYL5); chromosome X open readingframe 61 (CXorf61); hematopoietically expressed homeobox (HHEX); cAMPresponsive element binding protein 3-like4 (CREB3L4); X-box bindingprotein 1 (XBP1); SAM pointed domain containing ets trsanscriptionfactor (SPDEF); nuclear receptor coactivator 7 (NCOA7); galaninprepropeptide (GAL); HECT and RLD domain containing E3 ubiquitin proteinligase 5 (HERC5); major histocompatibility complex, class I, A (HLA-A);centromere protein V (CENPV); frequently rearranged in advanced T-celllymphomas 2 (FRAT2); phospholipase B domain containing 1 (PLBD1);adenosine A2b receptor (ADORA2B); G protein-coupled receptor, family C,group 5, member A (GPRC5A); enoyl CoA hydratase domain containing 1(ECHDC1); guanylate binding protein 1, interferon-inducible (GBP1);sulfatase 2 (SULF2), uncharacterized LOC100507463 (LOC100507463), andKIAA1324 (KIAA1324).

Of the 58 genes above, the following high expression genes are relatedto the “sensitive” signature: TGFB3, CYP4Z2P, ERBB2, DCXR, ABCC11, TRIL,LOC440335, INHBB, C17orf28, LIMK2, LARGE, SLC40A1, GPR160, CISH, PLCB4,BLNK, PRODH, RHOB, CREB3L4, XBP1, SPDEF, FRAT2, and KIAA1324; and thefollowing low expression genes are related to the “sensitive” signature:CD44, IFI44, SLC9A6, LYN, PHLDA1, PPARG, UPP1, AKR1C2, BAG2, DKK1, IRS2,IFIT1, FMNL2, LIF, TGFBR2, PLCG2, CAV2, IFIT3, CALB2, TSPYL5, CXorf61,HHEX, NCOA7, GAL, HERC5, HLA-A, CENPV, PLBD1, ADORA2B, GPRC5A, ECHDC1,GBP1, SULF2, and LOC100507463.

Of the 58 genes above, the following low expression genes are related tothe “resistant” signature: TGFB3, CYP4Z2P, ERBB2, DCXR, ABCC11, TRIL,LOC440335, INHBB, C17orf28, LIMK2, LARGE, SLC40A1, GPR160, CISH, PLCB4,BLNK, PRODH, RHOB, CREB3L4, XBP1, SPDEF, FRAT2, and KIAA1324; and thefollowing high expression genes are related to the “resistant”signature: CD44, IFI44, SLC9A6, LYN, PHLDA1, PPARG, UPP1, AKR1C2, BAG2,DKK1, IRS2, IFIT1, FMNL2, LIF, TGFBR2, PLCG2, CAV2, IFIT3, CALB2,TSPYL5, CXorf61, HHEX, NCOA7, GAL, HERC5, HLA-A, CENPV, PLBD1, ADORA2B,GPRC5A, ECHDC1, GBP1, SULF2, and LOC100507463.

This 58 gene signature prediction model may then be used to predict thesensitivity of breast tumor tissue samples from patients in order todetermine the tumor's sensitivity or resistance to treatment with anHDAC inhibitor.

The sensitive group of breast tumor tissues was enriched for threeclinical breast cancer diagnosis markers: ER positive, PR positive, andlow grade breast tumors. The resistant group of breat tumor tissues wasassociated with a triple negative (ER−PR−Her2/neu−) marker. Likewise,these three clinical breast cancer diagnosis markers may be used topredict the sensitivity of breast tumor tissue samples from patients inorder to determine the tumor's sensitivity or resistance to treatmentwith an HDAC inhibitor.

Histone Deacetylase (HDAC) Inhibitors

An HDAC inhibitor used in the methods of the invention can be any HDACinhibitor, such as a small molecule organic compound, an antibody, asiRNA, an aptamer, a nucleic acid, a protein, or a peptide. Preferably,the HDAC inhibitor is a small molecule organic compound.

Preferably, the HDAC inhibitor is an HDAC6 inhibitor. This means thatthe HDAC inhibitor selectively inhibits HDAC6 over other forms of HDAC.

In one aspect, the HDAC6 inhibitor is a compound of formula I:

or a pharmaceutically acceptable salt, ester or prodrug thereof,

wherein,

Z is N or CR*, wherein R* is an optionally substituted alkyl, anoptionally substituted acyl, an optionally substituted aryl or anoptionally substituted heteroaryl;

ring A is an optionally substituted aryl or an optionally substitutedheteroaryl;

ring B is an optionally substituted aryl or an optionally substitutedheteroaryl;

R₁ is (i) H, alkyl, haloalkyl, alkenyl, aryl, arylalkyl, heteroaryl,heterocyclic, carbocyclic, C(O)—R₂, C(O)O—R₂, or S(O)_(p), each of whichmay be optionally substituted; or

(ii) when Z is CR*, R₁ may be optionally substituted branched alkyl,OR₃, or N(R₃)(R₃), —CH₂CH₂OH, OCH₂CH₂OH, SH, or thio alkoxy;

or ring B and R₁ may together with the atom to which each is attached,form an optionally substituted heterocyclic, or an optionallysubstituted heteroaryl;

or R* and R₁ together with the atom to which each is attached, may forman optionally substituted carbocyclic, optionally substitutedheterocyclic, optionally substituted aryl or optionally substitutedheteroaryl ring;

R is H or an optionally substituted alkyl; or R and ring A may be joinedto form a fused bicyclic ring which may be optionally substituted;

each R₂ is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, orheteroaryl, each of which is optionally substituted;

each R₃ is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, orheteroaryl, each of which is optionally substituted;

n is 4, 5, 6, 7 or 8; and

p is 0, 1, or 2.

In one embodiment, the ring A is phenyl, naphthyl, anthracenyl,pyridinyl, pyrimidinyl, pyrazinyl, indolyl, imidazolyl, oxazolyl, furyl,thienyl, thiazolyl, triazolyl, isoxazolyl, quinolinyl, pyrrolyl,pyrazolyl, or 5,6,7,8-tetrahydroisoquinoline; each of which may beoptionally substituted.

In another embodiment, the ring B is phenyl, naphthyl, anthracenyl,pyridinyl, pyrimidinyl, pyrazinyl, indolyl, imidazolyl, oxazolyl, furyl,thienyl, thiazolyl, triazolyl, isoxazolyl, quinolinyl, pyrrolyl,pyrazolyl, or 5,6,7,8-tetrahydroisoquinoline; each of which may beoptionally substituted.

In certain embodiments, R₁ is H, optionally substituted alkyl,optionally substituted aryl, or optionally substituted heteroaryl, or R₁is OH or alkoxy.

In a further embodiment, R₁ is H, methyl, ethyl, propyl, i-propyl,butyl, i-butyl, t-butyl, pentyl, hexyl, phenyl, naphthyl, pyridinyl, OH,OCH₃, OCH₂CH₃, O-Pr, O-iPr, O-Bu, O-sBu, or O-tBu; each of which may beoptionally substituted.

In various embodiments, R₁ is OH, alkoxy, NH₂, NH(alkyl),N(alkyl)(alkyl), NH-aryl, NH-hetroaryl, N(aryl)(aryl),N(aryl)(heteroaryl), or N(heteroaryl)(heteroaryl).

In other embodiments, the carbonyl and the Z group attached to ring Aare disposed para to each other.

In other embodiments, the carbonyl and Z group attached to ring A aredisposed meta to each other.

In another embodiment, the carbonyl and the Z group attached to ring Aare disposed ortho to each other.

In one embodiment, the HDAC6 inhibitor is a compound formula II:

or a pharmaceutically acceptable salt, ester or prodrug thereof,

wherein,

each of X₁, X₂, X₃, or X₄ is independently N, CR′, O, S, NCR′, CR′ CR′,OCR′, SCR′, or absent, or X₁ or X₄ may be joined with R to form abicyclic ring; wherein up to three of X₁, X₂, X₃, or X₄ may be N;

ring B is an optionally substituted aryl or an optionally substitutedheteroaryl;

R₁ is H, alkyl, haloalkyl, alkenyl, aryl, arylalkyl, heteroaryl,heterocyclic, carbocyclic, C(O)—R₂, or C(O)O—R₂, each of which may beoptionally substituted;

R is H or an optionally substituted alkyl; or R and X₁ or X₄ may bejoined to form a fused bicyclic ring which may be optionallysubstituted;

each R′ is independently H, optionally substituted alkyl, halo, OH, NH₂,NHR″, haloalkyl, CN, N₃, NO₂;

R″ is H or alkyl; and

R₂ is alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each ofwhich is optionally substituted.

In certain embodiments, X₁, X₂, X₃, and X₄ are all CR′.

In other embodiments, X₂ and X₃, are N and X₁ and X₄ are CR′.

In another embodiment, X₂ and X₃, are CR′ and X₁ and X₄ are N.

In still other embodiments, X₂, is N; X₃ is S, N or O; X₁ is CR′ and X₄is absent.

In one embodiment, ring B is phenyl, pyridinyl, pyrimidinyl, orpyrazinyl; each of which may be optionally substituted.

In a further embodiment, ring B is substituted by alkyl, aryl,heteroaryl, cycloalkyl, heterocycloalkyl, aralkyl, haloalkyl, hal, OH,NH₂, NHR″, CN, N₃, or NO₂.

In certain embodiments, R₁ is H, alkyl, aryl, arylalkyl, or heteroaryl,each of which may be optionally substituted.

In another embodiment, the HDAC6 inhibitor is a compound of formula III:

or a pharmaceutically acceptable salt, ester or prodrug thereof,

wherein,

ring B is an optionally substituted aryl or an optionally substitutedheteroaryl;

R₁ is H, alkyl, haloalkyl, alkenyl, aryl, arylalkyl, heteroaryl,heterocyclic, carbocyclic, C(O)—R₂, or C(O)O—R₂, each of which may beoptionally substituted;

R₂ is optionally substituted heteroaryl, and

R is H or an optionally substituted alkyl; or R and the phenyl ring maybe joined to form a fused [6,5]bicyclic ring which may be optionallysubstituted.

In one embodiment, ring B is phenyl, pyridinyl, pyrimidinyl, orpyrazinyl; each of which may be optionally substituted.

In a further embodiment, ring B is substituted by alkyl, aryl, aralkyl,haloalkyl, hal, OH, NH₂, CN, or NO₂.

In other embodiments, R₁ is H, alkyl, aryl, arylalkyl, heteroaryl,C(O)—R₂, or C(O)O—R₂, each of which may be optionally substituted.

In various embodiments, R₂ is optionally substituted pyridinyl.

In another embodiment, the HDAC6 inhibitor is a compound of formula IV:

or a pharmaceutically acceptable salt, ester or prodrug thereof,

wherein,

ring B is an optionally substituted aryl or an optionally substitutedheteroaryl;

R₁ is H, alkyl, haloalkyl, alkenyl, aryl, arylalkyl, heteroaryl,heterocyclic, or carbocyclic, each of which may be optionallysubstituted;

or ring B and R₁ may together with the atom to which each is attached,form an optionally substituted heterocyclic, or an optionallysubstituted heteroaryl, and

R is H or an optionally substituted alkyl; or R and the 1,3-pyrimidinylring may be joined to form a fused bicyclic ring which may be optionallysubstituted.

In certain embodiments, ring B is phenyl, pyridinyl, pyrimidinyl, orpyrazinyl; each of which may be optionally substituted.

In a further embodiment, ring B is substituted by alkyl, aryl, aralkyl,haloalkyl, halo, OH, NH₂, CN, or NO₂.

In other embodiments, R₁ is H, alkyl, aryl, arylalkyl, or heteroaryl,each of which may be optionally substituted.

In another embodiment, R₁ is substituted by OH or halo.

In certain embodiments, the ring formed by ring B and R₁ is piperidine,pyrrolidine, tetrahydroquinoline, morpholine, piperazine,tetrahydro-triazolo pyrazine, or diazepane, each of which is optionallysubstituted.

In another embodiment, the HDAC6 inhibitor is a compound of formula V:

or a pharmaceutically acceptable salt, ester or prodrug thereof,

wherein,

each of X₁, X₂, or X₃ is independently N or CR′;

ring B is an optionally substituted aryl or an optionally substitutedheteroaryl;

R₁ is H, alkyl, haloalkyl, alkenyl, aryl, arylalkyl, heteroaryl,heterocyclic, or carbocyclic, each of which may be optionallysubstituted;

each R_(A) and R_(B) is independently H, NH(R_(C)), N(R_(C))(R_(C)),N(R_(C))CO(R_(C)), CO₂H, C(O)R_(C), C(O)OR_(C), C(O)NH₂, C(O)NH(R_(C)),C(O)N(R_(C))(R_(C)), SO₂R_(C), SOR_(C), SR_(C), alkyl, aryl, arylalkyl,alkoxy, heteroaryl, heterocyclic, and carbocyclic, each of which may befurther substituted; or R_(A) and R_(B) together with the carbon towhich they are attached form a carbonyl;

each R_(C) is independently H, alkyl, alkenyl, aryl, heteroaryl,cycloalkyl, or heterocyclic, each of which may be further substituted;

R′ is H, optionally substituted alkyl, halo, OH, NH₂, NHR″, haloalkyl,CN, N₃, NO₂;

R″ is H or alkyl; and

m is 1 or 2.

In a related embodiment, the HDAC6 inhibitor is a compound of formulaVa:

or a pharmaceutically acceptable salt, ester or prodrug thereof,

wherein,

each of X₁, X₂, or X₃ is independently N or CR′;

ring B is an optionally substituted aryl or an optionally substitutedheteroaryl;

R₁ is H, alkyl, haloalkyl, alkenyl, aryl, arylalkyl, heteroaryl,heterocyclic, or carbocyclic, each of which may be optionallysubstituted;

each R_(A) and R_(B) is independently H, NH(R_(C)), N(R_(C))(R_(C)),N(R_(C))CO(R_(C)), CO₂H, C(O)R_(C), C(O)OR_(C), C(O)NH₂, C(O)NH(R_(C)),C(O)N(R_(C))(R_(C)), SO₂R_(C), SOR_(C), SR_(C), alkyl, aryl, arylalkyl,alkoxy, heteroaryl, heterocyclic, and carbocyclic, each of which may befurther substituted; or R_(A) and R_(B) together with the carbon towhich they are attached form a carbonyl;

each R_(C) is independently H, alkyl, alkenyl, aryl, heteroaryl,cycloalkyl, or heterocyclic, each of which may be further substituted;

R′ is H, optionally substituted alkyl, halo, OH, NH₂, NHR″, haloalkyl,CN, N₃, NO₂;

R″ is H or alkyl; and

m is 1 or 2.

In one embodiment, X₁, X₂, and X₃, are all independently CR′.

In another embodiment, ring B is phenyl, pyridinyl, pyrimidinyl, orpyrazinyl; each of which may be optionally substituted.

In a further embodiment, ring B is substituted by alkyl, aryl,heteroaryl, cycloalkyl, heterocycloalkyl, aralkyl, haloalkyl, halo, OH,NH₂, NHR″, CN, N₃, or NO₂.

In certain embodiments, R₁ is H, alkyl, aryl, arylalkyl, or heteroaryl,each of which may be optionally substituted.

In another embodiment, the HDAC6 inhibitor is a compound of formula VI:

or a pharmaceutically acceptable salt, ester or prodrug thereof,

wherein,

ring B is an optionally substituted aryl or an optionally substitutedheteroaryl;

R* is an optionally substituted alkyl, an optionally substituted aryl oran optionally substituted heteroaryl;

R₁ is H, alkyl, aryl, arylalkyl, heteroaryl, heterocyclic, carbocyclic,OH, alkoxy, NH₂, NH(alkyl), or N(alkyl)(alkyl);

or R* and R₁ together with the atom to which each is attached, may forman optionally substituted carbocyclic, optionally substitutedheterocyclic, optionally substituted aryl or optionally substitutedheteroaryl ring; and

R is H or an optionally substituted alkyl.

In one embodiment, ring B is phenyl, pyridinyl, pyrimidinyl, pyrazinyl,or thiazole; each of which may be optionally substituted.

In another embodiment, R* is methyl, trifluoromethyl, phenyl, pyridinyl,pyrimidinyl, pyrazinyl, or thiazole; each of which may be optionallysubstituted.

In certain embodiments, R₁ is OH, methoxy, or ethoxy.

In various embodiments, ring B and R* are each independently substitutedwith one or more of alkyl, halogen, or C(O)NR_(X)R_(Y), wherein R_(X) isH or alkyl, and R_(Y) is H or alkyl.

In other embodiments, ring B and R* are each independently substitutedwith one or more of methyl, F, or C(O)N(Me)₂.

Representative compounds of the invention include, but are not limitedto, the following compounds of Table 1 below.

TABLE 1

In a most preferred embodiment of the invention, the HDAC6 inhibitor hasthe following chemical structure:

which is referred to herein as Compound A.

In preferred embodiments, a compound useful in the methods of theinvention has one or more of the following properties: the compound iscapable of inhibiting at least one histone deacetylase; the compound iscapable of inhibiting HDAC6; the compound is a selective HDAC6inhibitor; the compound binds to the poly-ubiquitin binding domain ofHDAC6; the compound is capable of inducing apoptosis in cancer cells(especially multiple myeloma cells, non-Hodgkin's lymphoma (NML) cells,breast cancer cells, acute myelogeous leukemia (AML) cells); and/or thecompound is capable of inhibiting aggresome formation.

In certain preferred embodiments, a compound useful in the methods ofthe invention comprises a metal binding moiety, preferably azinc-binding moiety such as a hydroxamate. As noted above, certainhydroxamates are potent inhibitors of HDAC6 activity; without wishing tobe bound by theory, it is believed that the potency of thesehydroxamates is due, at least in part, to the ability of the compoundsto bind zinc. In preferred embodiments, a compound useful in the methodsof the invention includes at least one portion or region which canconfer selectivity for a biological target implicated in the aggresomepathway, e.g., a biological target having tubulin deacetylase (TDAC) orHDAC activity, e.g., HDAC6. Thus, in certain preferred embodiments, acompound useful in the methods of the invention includes a zinc-bindingmoiety spaced from other portions of the molecule which are responsiblefor binding to the biological target.

The invention also provides for a pharmaceutical composition comprisinga compound of formula I, or a pharmaceutically acceptable ester, salt,or prodrug thereof, together with a pharmaceutically acceptable carrier.

Another object of the present invention is the use of a compound asdescribed herein (e.g., of any formulae herein) in the manufacture of amedicament for use in the treatment of a disorder or disease herein.Another object of the present invention is the use of a compound asdescribed herein (e.g., of any formulae herein) for use in the treatmentof a disorder or disease herein.

A compound useful in the methods of the invention can be prepared as apharmaceutically acceptable acid addition salt by reacting the free baseform of the compound with a pharmaceutically acceptable inorganic ororganic acid. Alternatively, a pharmaceutically acceptable base additionsalt of a compound of the invention can be prepared by reacting the freeacid form of the compound with a pharmaceutically acceptable inorganicor organic base.

Alternatively, the salt forms of the compounds useful in the methods ofthe invention can be prepared using salts of the starting materials orintermediates.

The free acid or free base forms of the compounds useful in the methodsof the invention can be prepared from the corresponding base additionsalt or acid addition salt from, respectively. For example a compounduseful in the methods of the invention in an acid addition salt form canbe converted to the corresponding free base by treating with a suitablebase (e.g., ammonium hydroxide solution, sodium hydroxide, and thelike). A compound useful in the methods of the invention in a baseaddition salt form can be converted to the corresponding free acid bytreating with a suitable acid (e.g., hydrochloric acid, etc.).

Compounds useful in the methods of the present invention can beconveniently prepared, or formed during the process described herein, assolvates (e.g., hydrates). Hydrates of compounds useful in the methodsof the present invention can be conveniently prepared byrecrystallization from an aqueous/organic solvent mixture, using organicsolvents such as dioxan, tetrahydrofuran or methanol.

In addition, some of the compounds useful in the methods of thisinvention have one or more double bonds, or one or more asymmetriccenters. Such compounds can occur as racemates, racemic mixtures, singleenantiomers, individual diastereomers, diastereomeric mixtures, and cis-or trans- or E- or Z-double isomeric forms, and other stereoisomericforms that may be defined, in terms of absolute stereochemistry, as (R)—or (S)—, or as (D)- or (L)- for amino acids. All such isomeric forms ofthese compounds are expressly included in the present invention. Opticalisomers may be prepared from their respective optically activeprecursors by the procedures described above, or by resolving theracemic mixtures. The resolution can be carried out in the presence of aresolving agent, by chromatography or by repeated crystallization or bysome combination of these techniques which are known to those skilled inthe art. Further details regarding resolutions can be found in Jacques,et al., Enantiomers, Racemates, and Resolutions (John Wiley & Sons,1981). The compounds useful in the methods of this invention may also berepresented in multiple tautomeric forms, in such instances, theinvention expressly includes all tautomeric forms of the compoundsdescribed herein. When the compounds described herein contain olefinicdouble bonds or other centers of geometric asymmetry, and unlessspecified otherwise, it is intended that the compounds include both Eand Z geometric isomers. Likewise, all tautomeric forms are alsointended to be included. The configuration of any carbon-carbon doublebond appearing herein is selected for convenience only and is notintended to designate a particular configuration unless the text sostates; thus a carbon-carbon double bond depicted arbitrarily herein astrans may be cis, trans, or a mixture of the two in any proportion. Allsuch isomeric forms of such compounds are expressly included in themethods of the present invention. All crystal forms of the compoundsdescribed herein are expressly included in the methods of the presentinvention.

The compounds useful in the methods of the invention are defined hereinby their chemical structures and/or chemical names. Where a compound isreferred to by both a chemical structure and a chemical name, and thechemical structure and chemical name conflict, the chemical structure isdeterminative of the compound's identity.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable herein includes that embodiment as any single embodimentor in combination with any other embodiments or portions thereof.

Other Cancer Treatments

Any cancer treatment, such as a chemotherapy drug or other anti-cancerdrug, may be used in combination with an HDAC inhibitor in the methodsof the invention. The chemotherapy drug or other anti-cancer drug maybe, for example, a small molecule organic compound, an antibody, asiRNA, an aptamer, a nucleic acid, a protein, or a peptide. Preferably,the anti-cancer drug is a proteasome inhibitor. More preferably, theproteasome inhibitor is bortezomib.

Pharmaceutical Compositions

In another aspect, the HDAC inhibitor is provided in a pharmaceuticalcomposition comprising a compound of formula I, or a pharmaceuticallyacceptable ester, salt, or prodrug thereof, together with apharmaceutically acceptable carrier. In a further aspect, the othercancer therapy is provided in a pharmaceutical composition.

Compounds useful in the methods of the invention can be administered aspharmaceutical compositions by any conventional route, in particularenterally, e.g., orally, e.g., in the form of tablets or capsules, orparenterally, e.g., in the form of injectable solutions or suspensions,topically, e.g., in the form of lotions, gels, ointments or creams, orin a nasal or suppository form. Pharmaceutical compositions comprising acompound useful in the methods of the present invention in free form orin a pharmaceutically acceptable salt form in association with at leastone pharmaceutically acceptable carrier or diluent can be manufacturedin a conventional manner by mixing, granulating or coating methods. Forexample, oral compositions can be tablets or gelatin capsules comprisingthe active ingredient together with a) diluents, e.g., lactose,dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; b)lubricants, e.g., silica, talcum, stearic acid, its magnesium or calciumsalt and/or polyethyleneglycol; for tablets also c) binders, e.g.,magnesium aluminum silicate, starch paste, gelatin, tragacanth,methylcellulose, sodium carboxymethylcellulose and orpolyvinylpyrrolidone; if desired d) disintegrants, e.g., starches, agar,alginic acid or its sodium salt, or effervescent mixtures; and/or e)absorbents, colorants, flavors and sweeteners. Injectable compositionscan be aqueous isotonic solutions or suspensions, and suppositories canbe prepared from fatty emulsions or suspensions. The compositions may besterilized and/or contain adjuvants, such as preserving, stabilizing,wetting or emulsifying agents, solution promoters, salts for regulatingthe osmotic pressure and/or buffers. In addition, they may also containother therapeutically valuable substances. Suitable formulations fortransdermal applications include an effective amount of a compound ofthe present invention with a carrier. A carrier can include absorbablepharmacologically acceptable solvents to assist passage through the skinof the host. For example, transdermal devices are in the form of abandage comprising a backing member, a reservoir containing the compoundoptionally with carriers, optionally a rate controlling barrier todeliver the compound to the skin of the host at a controlled andpredetermined rate over a prolonged period of time, and means to securethe device to the skin. Matrix transdermal formulations may also beused. Suitable formulations for topical application, e.g., to the skinand eyes, are preferably aqueous solutions, ointments, creams or gelswell-known in the art. Such may contain solubilizers, stabilizers,tonicity enhancing agents, buffers and preservatives.

Compounds useful in the methods of the invention can be administered intherapeutically effective amounts in combination with one or moretherapeutic agents (pharmaceutical combinations). For example,synergistic effects can occur with other anti-proliferative,anti-cancer, immunomodulatory or anti-inflammatory substances. Where thecompounds of the invention are administered in conjunction with othertherapies, dosages of the co-administered compounds will of course varydepending on the type of co-drug employed, on the specific drugemployed, on the condition being treated and so forth.

Combination therapy includes the administration of the subject compoundsin further combination with other biologically active ingredients (suchas, but not limited to, a second and different antineoplastic agent) andnon-drug therapies (such as, but not limited to, surgery or radiationtreatment). For instance, the compounds of the invention can be used incombination with other pharmaceutically active compounds, preferablycompounds that are able to enhance the effect of the compounds of theinvention. The compounds of the invention can be administeredsimultaneously (as a single preparation or separate preparation) orsequentially to the other drug therapy. In general, a combinationtherapy envisions administration of two or more drugs during a singlecycle or course of therapy.

In certain embodiments, these compositions optionally further compriseone or more additional therapeutic agents. Alternatively, a compounduseful in the methods of this invention may be administered to a patientin need thereof in combination with the administration of one or moreother therapeutic agents. For example, additional therapeutic agents forconjoint administration or inclusion in a pharmaceutical compositionwith a compound useful in the methods of this invention may be anapproved chemotherapeutic agent, or it may be any one of a number ofagents undergoing approval in the Food and Drug Administration thatultimately obtains approval for the treatment of any disorder associatedwith cellular hyperproliferation. In certain other embodiments, theadditional therapeutic agent is an anticancer agent, as discussed inmore detail herein. In the treatment of cancer or protein degradationdisorders, the compound may be combined with a proteasome inhibitor(e.g., bortezomib, R1 15777 FTI, MG132, NPI-0052, etc.). In thetreatment of cancer or protein degradation disorders, the compound maybe combined with protein degradation inhibitor (e.g. another inventivecompound, a tubacin-like compound, bortezomib, R1 15777 FTI, MG132,NPI-0052, SAHA, ¹⁶⁶Ho-DOTMP, arsenic trioxide, 17-AAG, MG 132, etc.).

It will also be appreciated that the compounds and pharmaceuticalcompositions useful in the methods of the present invention can beemployed in combination therapies, that is, the compounds andpharmaceutical compositions can be administered concurrently with, priorto, or subsequent to, one or more other desired therapeutics or medicalprocedures. The particular combination of therapies (therapeutics orprocedures) to employ in a combination regimen will take into accountcompatibility of the desired therapeutics and/or procedures and thedesired therapeutic effect to be achieved. It will also be appreciatedthat the therapies employed may achieve a desired effect for the samedisorder (for example, an inventive compound may be administeredconcurrently with another anticancer agent), or they may achievedifferent effects (e.g., control of any adverse effects).

It will also be appreciated that the compounds and pharmaceuticalcompositions useful in the methods of the present invention can beformulated and employed in combination therapies, that is, the compoundsand pharmaceutical compositions can be formulated with or administeredconcurrently with, prior to, or subsequent to, one or more other desiredtherapeutics or medical procedures. The particular combination oftherapies (therapeutics or procedures) to employ in a combinationregimen will take into account compatibility of the desired therapeuticsand/or procedures and the desired therapeutic effect to be achieved. Itwill also be appreciated that the therapies employed may achieve adesired effect for the same disorder (for example, an inventive compoundmay be administered concurrently with another immunomodulatory agent,anticancer agent or agent useful for the treatment of psoriasis), orthey may achieve different effects (e.g., control of any adverseeffects).

For example, other therapies or anticancer agents that may be used incombination with the compounds useful in the methods of the presentinvention include, but not limited to, surgery, radiotherapy (in but afew examples, gamma-radiation, neutron beam radiotherapy, electron beamradiotherapy, proton therapy, brachytherapy, and systemic radioactiveisotopes, to name a few), endocrine therapy, biologic response modifiers(interferons, interleukins, antibodies, aptamers, siRNAs,oligonucletoides, enzyme, ion channel and receptor inhibitors oractivators to name a-few), hyperthermia and cryotherapy, agents toattenuate any adverse effects (e.g., antiemetics), and other approvedchemotherapeutic drugs, including, but not limited to, alkylating drugs(e.g., mechlorethamine, chlorambucil, Cyclophosphamide, Melphalan,Ifosfamide), antimetabolites (e.g., Methotrexate), purine antagonistsand pyrimidine antagonists (e.g., 6-Mercaptopurine, 5-Fluorouracil,Cytarabile, Gemcitabine), spindle poisons (e.g., Vinblastine,Vincristine, Vinorelbine, Paclitaxel), podophyllotoxins (e.g.,Etoposide, Irinotecan, Topotecan), antibiotics (Doxorubicin, Bleomycin,Mitomycin), nitrosoureas (e.g., Carmustine, Lomustine), inorganic ions(e.g., Cisplatin, Carboplatin), enzymes (e.g., Asparaginase), andhormones (e.g., Tamoxifen, Leuprolide, Flutamide, and Megestrol), toname a few. For a more comprehensive discussion of updated cancertherapies see, The Merck Manual, Seventeenth Ed. 1999, the entirecontents of which are hereby incorporated by reference. See also theNational Cancer Institute (CNI) website (www dot nci dot nih dot gov)and the Food and Drug Administration (FDA) website for a list of the FDAapproved oncology drugs (www dot fda dot gov/cder/cancer/dmglistfrarne).

In certain embodiments, the pharmaceutical compositions useful in themethods of the present invention further comprise one or more additionaltherapeutically active ingredients (e.g., chemotherapeutic and/orpalliative). For purposes of the invention, the term “palliative” refersto treatment that is focused on the relief of symptoms of a diseaseand/or side effects of a therapeutic regimen, but is not curative. Forexample, palliative treatment encompasses painkillers, antinauseamedications, anti-pyretics, and anti-sickness drugs. In addition,chemotherapy, radiotherapy and surgery can all be used palliatively(that is, to reduce symptoms without going for cure; e.g., for shrinkingtumors and reducing pressure, bleeding, pain and other symptoms ofcancer).

The compounds and compositions can be administered together withhormonal and steroidal anti-inflammatory agents, such as but not limitedto, estradiol, conjugated estrogens (e.g., PREMARIN, PREMPRO, ANDPREMPHASE), 17 beta estradiol, calcitonin-salmon, levothyroxine,dexamethasone, medroxyprogesterone, prednisone, cortisone, flunisolide,and hydrocortisone; non-steroidal anti-inflammatory agents, such as butnot limited to, tramadol, fentanyl, metamizole, ketoprofen, naproxen,nabumetone, ketoralac, tromethamine, loxoprofen, ibuprofen, aspirin, andacetaminophen; anti-TNF-α antibodies, such as infliximab (REMICADE™) andetanercept (ENBREL™).

The pharmaceutical compositions useful in the methods of the presentinvention comprise a therapeutically effective amount of a compound ofthe present invention formulated together with one or morepharmaceutically acceptable carriers. As used herein, the term“pharmaceutically acceptable carrier” means a non-toxic, inert solid,semi-solid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. The pharmaceutical compositionsuseful in the methods of this invention can be administered to humansand other animals orally, rectally, parenterally, intracisternally,intravaginally, intraperitoneally, topically (as by powders, ointments,or drops), buccally, or as an oral or nasal spray.

According to the methods of treatment of the present invention,disorders are treated or prevented in a subject, such as a human orother animal, by administering to the subject a therapeuticallyeffective amount of a compound of the invention, in such amounts and forsuch time as is necessary to achieve the desired result. The term“therapeutically effective amount” of a compound useful in the methodsof the invention, as used herein, means a sufficient amount of thecompound so as to decrease the symptoms of a disorder in a subject. Asis well understood in the medical arts a therapeutically effectiveamount of a compound of this invention will be at a reasonablebenefit/risk ratio applicable to any medical treatment.

In general, compounds useful in the methods of the invention will beadministered in therapeutically effective amounts via any of the usualand acceptable modes known in the art, either singly or in combinationwith one or more therapeutic agents. A therapeutically effective amountmay vary widely depending on the severity of the disease, the age andrelative health of the subject, the potency of the compound used andother factors. In general, satisfactory results are indicated to beobtained systemically at daily dosages of from about 0.03 to 2.5 mg/kgper body weight (0.05 to 4.5 mg/m²). An indicated daily dosage in thelarger mammal, e.g. humans, is in the range from about 0.5 mg to about100 mg, conveniently administered, e.g. in divided doses up to fourtimes a day or in retard form. Suitable unit dosage forms for oraladministration comprise from ca. 1 to 50 mg active ingredient.

In certain embodiments, a therapeutic amount or dose of the compoundsuseful in the methods of the present invention may range from about 0.1mg/kg to about 500 mg/kg (about 0.18 mg/m² to about 900 mg/m²),alternatively from about 1 to about 50 mg/kg (about 1.8 to about 90mg/m²). In general, treatment regimens according to the presentinvention comprise administration to a patient in need of such treatmentfrom about 10 mg to about 1000 mg of the compound(s) useful in themethods of this invention per day in single or multiple doses.Therapeutic amounts or doses will also vary depending on route ofadministration, as well as the possibility of co-usage with otheragents.

Upon improvement of a subject's condition, a maintenance dose of acompound, composition or combination useful in the methods of thisinvention may be administered, if necessary. Subsequently, the dosage orfrequency of administration, or both, may be reduced, as a function ofthe symptoms, to a level at which the improved condition is retainedwhen the symptoms have been alleviated to the desired level, treatmentshould cease. The subject may, however, require intermittent treatmenton a long-term basis upon any recurrence of disease symptoms.

It will be understood, however, that the total daily usage of thecompounds and compositions useful in the methods of the presentinvention will be decided by the attending physician within the scope ofsound medical judgment. The specific inhibitory dose for any particularpatient will depend upon a variety of factors including the disorderbeing treated and the severity of the disorder; the activity of thespecific compound employed; the specific composition employed; the age,body weight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed; andlike factors well known in the medical arts.

The terms “co-administration” or “combined administration” or the likeas utilized herein are meant to encompass administration of the selectedtherapeutic agents to a single patient, and are intended to includetreatment regimens in which the agents are not necessarily administeredby the same route of administration or at the same time.

The term “pharmaceutical combination” as used herein means a productthat results from the mixing or combining of more than one activeingredient and includes both fixed and non-fixed combinations of theactive ingredients. The term “fixed combination” means that the activeingredients, e.g., a compound useful in the methods of the invention anda co-agent, are both administered to a patient simultaneously in theform of a single entity or dosage. The term “non-fixed combination”means that the active ingredients, e.g., a compound useful in themethods of the invention and a co-agent, are both administered to apatient as separate entities either simultaneously, concurrently orsequentially with no specific time limits, wherein such administrationprovides therapeutically effective levels of the two compounds in thebody of the patient. The latter also applies to cocktail therapy, e.g.,the administration of three or more active ingredients.

Types of Cancer

The methods of the invention may be used to identify cancer patientsthat will or are likely to respond to treatment with an HDAC inhibitor,alone or in combination with another cancer therapy. The methods of theinvention may be used to identify patients having any type of cancer.Preferably, the cancer is selected from the group consisting of:brain/neuronal cancer, breast cancer, cancer of the central nervoussystem, haematopoietic and lymphoid tissue cancer, kidney cancer, cancerof the large intestine, liver cancer, lung cancer, cancer of theoesophagus, pancreatic cancer, prostate cancer, skin cancer, soft tissuecancer, and stomach cancer.

Biological Samples

The methods of the invention may involve taking a biological sample froma patient in order to determine the gene mutation status of the sampleor the gene expression level status of the sample. For example, thebiological sample may be a from a tumor tissue, tumor biopsy, wholeblood, blood serum, blood plasma, semen, urine, mucus, or other bodysample. In a preferred embodiment of the invention, the biologicalsample is blood serum, blood plasma, tumor tissue, or tumor biopsysample. Tumor tissue may be formalin-fixed paraffin embedded tumortissue or fresh frozen tumor tissue.

Biomarkers

The methods of the invention may be used to identify biomarkers that canbe used to identify cancer patients that will or are likely to respondto treatment with a HDAC inhibitor, alone or in combination with anothercancer treatment. For example, gene mutations that are correlated withcell sensitivities to one or more cancer drugs may be used asbiomarkers. In addition, gene expression levels of genes that arecorrelated with cell sensitivities to one or more cancer drugs may beused as biomarkers.

Preferably, the biomarkers are selected from the group consisting ofhuman epidermal growth factor receptor 2 (Her2); erythroblastic leukemiaviral oncogene homolog 2 (ERBB2); SMAD family member 4 (SMAD4);phosphatase and tensin homolog (PTEN); epidermal growth factor receptoroncogene (EGFR); histone-lysine N-methyltransferase (EZH2); SET domaincontaining 2 (SETD2); von Hippel-Lindau tumor suppressor (VHL); pterin-4alpha-carbinolamine dehydratase/dimerization cofactor of hepatocytenuclear factor 1 alpha (PCBD1); protein phosphatase 2, regulatorysubunit B, gamma isoform (PPP2R2C); neural precursor cell expressed,developmentally downregulated 4 (NEDD4); prolyl 4-hydroxylase, alphapolypeptide II (P4HA2); SLC2A4 regulator (SLC2A4RG); sulfatase 2(SULF2); lysosomal protein transmembrane 4 alpha (LAPTM4A);3′-phosphoadenosine 5′-phosphosulfate synthase 2 (PAPSS2); aldo-ketoreductase family 1, member C1 (dihydrodiol dehydrogenase 1; 20-alpha(3-alpha)-hydroxysteroid dehydrogenase) (AKR1C1); protein tyrosinephosphatase, non-receptor type 12 (PTPN12); DCN1, defective in cullinneddylation 1, domain containing 4 (S. cerevisiae) (DCUN1D4);ras-related C3 botulinum toxin substrate 2 (RAC2); acyl-Coexzyme Adehydrogenase, C-4 to C-112 straight chain (ACADM); Rho GTPaseactivating protein 4 (ARHGAP4); ATPase type 13A1 (ATP13A1); chemokinereceptor 7 (CCR7); coronin 7 (CORO7); CXXC finger 4 (CXXC4);differentially expressed in FDCP 6 homolog (DEF6); KRI1 homolog (KRI1);limb region 1 homolog (LMBR1L); leukotriene B4 receptor (LTB4R);RAD54-like 2 (RAD54L2); chromosome X open reading frame 21 (CXorf21);SREBF chaperone (SCAP); selectin L (SELL); splicing factor 3a, subunit 2(SF3A2); Lyrm7 homolog (LYRM7); O-linked N-acetylglucosamine transferase(OGT); tubulin, alpha 3c (TUBA3C); tubulin, alpha 3d (TUBA3D); KH-typesplicing regulatory protein (KHSRP); DEAH (Asp-Glu-Ala-His) boxpolypeptide 30 (DHX30); APEX nuclease (apurinic/apyrimidinicendonuclease) 2 (APEX2); abhydrolase domain containing 14A (ABHD14A);UDP-glucose dehydrogenase (UGDH); H2A histone family, member Y2(H2AFY2); myosin VC (MYO5C); nephronectin (NPNT); KIAA1598 (KIAA1598);serglycin (SRGN); collagen, type VI, alpha 3 (COL6A3); G-proteinsignaling modulator 3 (GPSM3); hydroxysteroid dehydrogenase 1 (HSD11B1);peroxisomal biogenesis factor 6 (PEX6); ras-related C3 botulinum toxinsubstrate 2 (RAC2); synovial sarcoma, X breakpoint 5 (SSX5); andacyl-Coenzyme A binding domain containing 3 (ACBD3). Each of these genesor gene mutations is well known in the art.

The invention also includes mutations, mutants, or variants of the abovebiomarker proteins or genes. In those mutations, mutants, or variants,the native sequence of the biomarker protein or gene is changed by oneor more substitutions, modifications, deletions, or insertions of one ormore amino acids or nucleic acids in the protein or gene. In addition,those mutations, mutants, or variants may also encompass geneamplification, chromosomal translocation, protein overexpression,protein underexpression, gene overexpression, and gene underexpression.The term “native sequence” refers to an amino acid sequence or a nucleicacid sequence that is identical to a wild-type or native form of abiomarker protein or gene.

Kits

Certain embodiments of the invention include kits that may be used inthe methods of the invention.

For example, in a certain embodiment of the invention, the inventionprovides a kit comprising a nucleic acid or probe that is complementaryto any one of the genes, or a fragment thereof, selected from the groupconsisting of erythroblastic leukemia viral oncogene homolog 2 (ERBB2);SMAD family member 4 (SMAD4); phosphatase and tensin homolog (PTEN);epidermal growth factor receptor oncogene (EGFR); histone-lysineN-methyltransferase (EZH2); SET domain containing 2 (SETD2); vonHippel-Lindau tumor suppressor (VHL); pterin-4 alpha-carbinolaminedehydratase/dimerization cofactor of hepatocyte nuclear factor 1 alpha(PCBD1); protein phosphatase 2, regulatory subunit B, gamma isoform(PPP2R2C); neural precursor cell expressed, developmentallydown-regulated 4 (NEDD4); prolyl 4-hydroxylase, alpha polypeptide II(P4HA2); SLC2A4 regulator (SLC2A4RG); sulfatase 2 (SULF2); lysosomalprotein transmembrane 4 alpha (LAPTM4A); 3′-phosphoadenosine5′-phosphosulfate synthase 2 (PAPSS2); aldo-keto reductase family 1,member C1 (dihydrodiol dehydrogenase 1; 20-alpha(3-alpha)-hydroxysteroid dehydrogenase) (AKR1C1); protein tyrosinephosphatase, non-receptor type 12 (PTPN12); DCN1, defective in cullinneddylation 1, domain containing 4 (S. cerevisiae) (DCUN1D4);ras-related C3 botulinum toxin substrate 2 (RAC2); acyl-Coexzyme Adehydrogenase, C-4 to C-112 straight chain (ACADM); Rho GTPaseactivating protein 4 (ARHGAP4); ATPase type 13A1 (ATP13A1); chemokinereceptor 7 (CCR7); coronin 7 (CORO7); CXXC finger 4 (CXXC4);differentially expressed in FDCP 6 homolog (DEF6); KRI1 homolog (KRI1);limb region 1 homolog (LMBR1L); leukotriene B4 receptor (LTB4R);RAD54-like 2 (RAD54L2); chromosome X open reading frame 21 (CXorf21);SREBF chaperone (SCAP); selectin L (SELL); splicing factor 3a, subunit 2(SF3A2); Lyrm7 homolog (LYRM7); O-linked N-acetylglucosamine transferase(OGT); tubulin, alpha 3c (TUBA3C); tubulin, alpha 3d (TUBA3D); KH-typesplicing regulatory protein (KHSRP); DEAH (Asp-Glu-Ala-His) boxpolypeptide 30 (DHX30); APEX nuclease (apurinic/apyrimidinicendonuclease) 2 (APEX2); abhydrolase domain containing 14A (ABHD14A);UDP-glucose dehydrogenase (UGDH); H2A histone family, member Y2(H2AFY2); myosin VC (MYO5C); nephronectin (NPNT); KIAA1598 (KIAA1598);serglycin (SRGN); collagen, type VI, alpha 3 (COL6A3); G-proteinsignaling modulator 3 (GPSM3); hydroxysteroid dehydrogenase 1 (HSD11B1);peroxisomal biogenesis factor 6 (PEX6); ras-related C3 botulinum toxinsubstrate 2 (RAC2); synovial sarcoma, X breakpoint 5 (SSX5); andacyl-Coenzyme A binding domain containing 3 (ACBD3), and instructionsfor use of the nucleic acid to detect the presence of the gene or theexpression level of the gene.

Another embodiment of the invention provides a kit comprising anantibody that binds to a protein produced by any one of the genes, or afragment thereof, selected from the group consisting of erythroblasticleukemia viral oncogene homolog 2 (ERBB2); SMAD family member 4 (SMAD4);phosphatase and tensin homolog (PTEN); epidermal growth factor receptoroncogene (EGFR); histone-lysine N-methyltransferase (EZH2); SET domaincontaining 2 (SETD2); von Hippel-Lindau tumor suppressor (VHL); pterin-4alpha-carbinolamine dehydratase/dimerization cofactor of hepatocytenuclear factor 1 alpha (PCBD1); protein phosphatase 2, regulatorysubunit B, gamma isoform (PPP2R2C); neural precursor cell expressed,developmentally downregulated 4 (NEDD4); prolyl 4-hydroxylase, alphapolypeptide II (P4HA2); SLC2A4 regulator (SLC2A4RG); sulfatase 2(SULF2); lysosomal protein transmembrane 4 alpha (LAPTM4A);3′-phosphoadenosine 5′-phosphosulfate synthase 2 (PAPSS2); aldo-ketoreductase family 1, member C1 (dihydrodiol dehydrogenase 1; 20-alpha(3-alpha)-hydroxysteroid dehydrogenase) (AKR1C1); protein tyrosinephosphatase, non-receptor type 12 (PTPN12); DCN1, defective in cullinneddylation 1, domain containing 4 (S. cerevisiae) (DCUN1D4);ras-related C3 botulinum toxin substrate 2 (RAC2); acyl-Coexzyme Adehydrogenase, C-4 to C-112 straight chain (ACADM); Rho GTPaseactivating protein 4 (ARHGAP4); ATPase type 13A1 (ATP13A1); chemokinereceptor 7 (CCR7); coronin 7 (CORO7); CXXC finger 4 (CXXC4);differentially expressed in FDCP 6 homolog (DEF6); KRI1 homolog (KRI1);limb region 1 homolog (LMBR1L); leukotriene B4 receptor (LTB4R);RAD54-like 2 (RAD54L2); chromosome X open reading frame 21 (CXorf21);SREBF chaperone (SCAP); selectin L (SELL); splicing factor 3a, subunit 2(SF3A2); Lyrm7 homolog (LYRM7); O-linked N-acetylglucosamine transferase(OGT); tubulin, alpha 3c (TUBA3C); tubulin, alpha 3d (TUBA3D); KH-typesplicing regulatory protein (KHSRP); DEAH (Asp-Glu-Ala-His) boxpolypeptide 30 (DHX30); APEX nuclease (apurinic/apyrimidinicendonuclease) 2 (APEX2); abhydrolase domain containing 14A (ABHD14A);UDP-glucose dehydrogenase (UGDH); H2A histone family, member Y2(H2AFY2); myosin VC (MYO5C); nephronectin (NPNT); KIAA1598 (KIAA1598);serglycin (SRGN); collagen, type VI, alpha 3 (COL6A3); G-proteinsignaling modulator 3 (GPSM3); hydroxysteroid dehydrogenase 1 (HSD11B1);peroxisomal biogenesis factor 6 (PEX6); ras-related C3 botulinum toxinsubstrate 2 (RAC2); synovial sarcoma, X breakpoint 5 (SSX5); andacyl-Coenzyme A binding domain containing 3 (ACBD3), and instructionsfor use of the antibody to detect the presence of the gene or theexpression level of the gene.

Pharmaceutical Combinations

Compounds of the invention can be administered in therapeuticallyeffective amounts in combination with one or more therapeutic agents(pharmaceutical combinations). In general, a combination therapyenvisions administration of two or more drugs during a single cycle orcourse of therapy. In a particular embodiment, the compounds of theinvention are administered in combination with lenalidomide (Revlimid),which has the following structure:

Revlimid is described, for example, in U.S. Pat. No. 5,635,517, which isincorporated herein by reference in its entirety.

It will also be appreciated that the compounds and pharmaceuticalcompositions of the present invention can be employed in combinationtherapies, that is, the compounds and pharmaceutical compositions can beadministered concurrently with, prior to, or subsequent to, one or moreother desired therapeutics or medical procedures. The particularcombination of therapies (therapeutics or procedures) to employ in acombination regimen will take into account compatibility of the desiredtherapeutics and/or procedures and the desired therapeutic effect to beachieved. It will also be appreciated that the therapies employed mayachieve a desired effect for the same disorder (for example, aninventive compound may be administered concurrently with anotheranticancer agent), or they may achieve different effects (e.g., controlof any adverse effects).

In an particular embodiment, provided herein is a pharmaceuticalcomposition comprising2-(diphenylamino)-N-(7-(hydroxyamino)-7-oxoheptyl)pyrimidine-5-carboxamide,Revlimid, and a pharmaceutically acceptable carrier. In anotherembodiment, provided herein is a method of treating multiple myeloma ina subject in need thereof comprising administering to the subject2-(diphenylamino)-N-(7-(hydroxyamino)-7-oxoheptyl)pyrimidine-5-carboxamide,and Revlimid. The agents can be administered together in one unit dose,separately but at approximately the same time, or at different times.

All patents, patent applications, and publications mentioned herein,both supra and infra, are hereby incorporated by reference in theirentirety.

EXAMPLES

The following examples are provided to aid in the understanding of theinvention. It is understood that modifications can be made to theprocedures set forth below without departing from the spirit and scopeof the invention.

Conventional techniques of molecular biology and nucleic acid chemistry,which are within the skill of the art, are explained in the literatureand used in the practice of the invention. See, for example, Sambrook,J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989; Gait, M. J. (ed.),Oligonucleotide synthesis—a practical approach, IRL Press Limited, 1984;Hames, B. D. and Higgins, S. J. (eds.), Nucleic acid hybridisation—apractical approach, IRL Press Limited, 1985; and a series, Methods inEnzymology, Academic Press, Inc., all of which are incorporated hereinby reference.

Example 1 Association Study of Cancer Types

This example describes the association study that was performed todetermine which types of cancer are sensitive to treatment with CompoundA alone or in combination with bortezomib.

65 cancer cell lines (including 12 of breast cancer, 11 of hematologiccancer, 9 of colorectal cancer, 6 of lung cancer, 6 of skin cancer, 4brain cancer, 3 of renal cancer, 3 of liver cancer, 3 of prostatecancer, 3 of pancreatic cancer, 3 of ovarian cancer, and 2 of stomachcancer) were selected for analysis. Each of the cell lines was culturedand treated with the HDAC6 inhibitor, Compound A, with or without theproteasome inhibitor, bortezomib, before their cell viabilities weremeasured at the end of treatments. The sensitivities of the cell lineswere described using the concentration of Compound A that provides 50%inhibition of cell viabilities (IC₅₀). In all analyses, the logarithm ofdrug sensitivity values (i.e., Log [IC50]) was used, which is morenormally distributed than the raw IC₅₀ values. This is because moststandard statistical tests (e.g., t-test and correlation analysis)assume the normal distribution of data points.

The results of this association study are as follows. Most cell lineswere resistant to treatment with either Compound A alone or bortezomibalone. Also, cell lines of ovarian cancer were resistant to combinationtreatment with Compound A and bortezomib. In addition, cell lines forbrain/neuron cancer, breast cancer, lymphoid cancer, kidney cancer,colon/large intestine cancer, and skin cancer were sensitive tocombination treatment with Compound A and bortezomib. Further, celllines for hematologic malignant cancer were the most sensitive totreatment with both Compound A alone, or combination treatment withCompound A and bortezomib.

Example 2 Association Study of Gene Mutations

This example describes the association study that was performed todetermine which gene mutations, and hence types of cancer, are sensitiveto treatment with a Compound A alone or in combination with bortezomib.

65 cancer cell lines (including 12 of breast cancer, 11 of hematologiccancer, 9 of colorectal cancer, 6 of lung cancer, 6 of skin cancer, 4brain cancer, 3 of renal cancer, 3 of liver cancer, 3 of prostatecancer, 3 of pancreatic cancer, 3 of ovarian cancer, and 2 of stomachcancer) were selected for analysis. Each of the cell lines was culturedand treated with the HDAC6 inhibitor, Compound A, with or without theproteasome inhibitor, bortezomib, before their cell viabilities weremeasured at the end of treatments. The sensitivities of the cell lineswere described using the concentration of Compound A that provides 50%inhibition of cell viabilities (IC₅₀). In all analyses, the logarithm ofdrug sensitivity values (i.e., Log [IC50]) was used, which is morenormally distributed than the raw IC₅₀ values. This is because moststandard statistical tests (e.g., t-test and correlation analysis)assume the normal distribution of data points.

Next, the genetic characteristics (in this example, mutations) of thetested cell lines associated with their drug responses wereinvestigated.

For the association tests of gene mutations, 50 common oncogene/tumorsuppressor genes were selected to analyze the results. Mutation profilesof the cell lines were downloaded from the Sanger COSMIC DB on theInternet: www dot sanger dot ac dot uk/cosmic. For each gene locus, the“Fisher's Exact Test” was used to calculate statistical significance(p<0.05) of association between the genotype and the cell sensitivitiesto the treatments. The genotypes of eight gene mutations were identifiedin these tests.

Overexpression of the human epidermal growth factor receptor 2 (Her2)protein, as compared to a normalized protein expression level of theprotein, caused by erythroblastic leukemia viral oncogene homolog 2(ERBB2) gene amplification or by any other means, in the breast cancercell lines was sensitive to treatment with Compound A.

Basal-type or triple negative mutations (estrogen receptor-negative,progesterone receptor-negative and HER2/neu-negative) in the breastcancer cell lines were resistant to treatment with Compound A.

A gene mutation in the SMAD4 gene in colorectal cancer cell lines wassensitive to combination treatment with Compound A and bortezomib.

A gene mutation in the phosphatase and tensin homolog gene, PTEN, in alltested cancer cell lines was sensitive to combination treatment withCompound A and bortezomib.

A gene mutation in the histone-lysine N-methyltransferase gene, EZH2, inall tested cancer cell lines was sensitive to combination treatment withCompound A and bortezomib.

A gene mutation in the epidermal growth factor receptor oncogene, EGFR,in all tested cancer cell lines was sensitive to combination treatmentwith Compound A and bortezomib.

A gene mutation in the SET domain containing 2 gene, SETD2, in alltested cancer cell lines was sensitive to combination treatment withCompound A and bortezomib.

A gene mutation in the Von Hippel-Lindau tumor suppressor gene, VHL, inall tested cancer cell lines was sensitive to combination treatment withCompound A and bortezomib.

Example 3 Association Study of Gene Expression Levels

This example describes the association study that was performed todetermine which gene expression profiles, and hence types of cancer, aresensitive to treatment with Compound A alone or in combination withbortezomib.

65 cancer cell lines (including 12 of breast cancer, 11 of hematologiccancer, 9 of colorectal cancer, 6 of lung cancer, 6 of skin cancer, 4brain cancer, 3 of renal cancer, 3 of liver cancer, 3 of prostatecancer, 3 of pancreatic cancer, 3 of ovarian cancer, and 2 of stomachcancer) were selected for analysis. Each of the cell lines was culturedand treated with the HDAC6 inhibitor, Compound A, with or without theproteasome inhibitor, bortezomib, before their cell viabilities weremeasured at the end of treatments. The sensitivities of the cell lineswere described using the concentration of Compound A that provides 50%inhibition of cell viabilities (IC₅₀). In all analyses, the logarithm ofdrug sensitivity values (i.e., Log [IC50]) was used, which is morenormally distributed than the raw IC₅₀ values. This is because moststandard statistical tests (e.g., t-test and correlation analysis)assume the normal distribution of data points.

Next, the genetic characteristics (in this example, gene expressionlevels) of the tested cell lines associated with their drug responseswere investigated.

For the association tests of gene expression levels, the gene-expressionprofiles of the cell lines were obtained from two independent sources:the ArrayExpress (AE) database on the Internet:<URL:http://www.ebi.ac.uk/arrayexpress/>, which is maintained by the EuropeanBioinformatics Institute, and the caArray database on the Internet:<URL:https://cabig.nci.nih gov/caArray_GSKdata/>, which is from NIH. Inaddition, the Affymetrix CEL files, which contain the expressionintensity of individual genes from these public databases, weredownloaded, and then the R/BioConductor packages on the Internet:<URL:http://bioconductor.org> were used in order to normalize thegene-expression values between individual cell lines. The correlationcoefficient and its significance (p<0.001) were calculated using theCORTEST function of R package between the IC₅₀ values and the normalizedgene expression levels for each probe-set. For validation, only geneswith all probe sets showing significant correlations were reported;genes were removed if any of its probe-sets were non-significant.

In total, the expression levels of 48 genes showed significantassociation with the cell sensitivities to the treatments.

Thirty-five of these forty-eight genes were associated withsensitivities to treatment with Compound A alone. Eleven genes, PCBD1,PPP2R2C, NEDD4, P4HA2, SLC2A4RG, SULF2, LAPTM4A, PAPSS2, AKR1C1, PTPN12,and DCUN1D4 had expression levels that were positively associated withthe IC₅₀ values (i.e., the higher the gene expression level, the moreresistant the cell is to the treatment; or the lower the gene expressionlevel, the more sensitive the cell is to the treatment). Twenty-fourgenes, RAC2, ACADM, ARHGAP4, ATP13A1, CCR7, CORO7, CXXC4, DEF6, KRI1,LMBR1L, LTB4R, RAD54L2, CXorf21, SCAP, SELL, SF3A2, LYRM7, OGT, TUBA3C,TUBA3D, KHSRP, DHX30, APEX2, and ABHD14A had expression levels that werenegatively associated with the IC₅₀ values (i.e., the higher the geneexpression level, the more sensitive the cell is to the treatment; orthe lower the gene expression level, the more resistant the cell is tothe treatment).

Thirteen of these forty-eight genes were associated with sensitivitiesto combination treatment with Compound A and bortezomib. Five genesUGDH, H2AFY2, MYO5C, NPNT, and KIAA1598, had expression levels that werepositively associated with the IC₅₀ values (i.e., the higher the geneexpression level, the more resistant the cell is to the treatment; orthe lower the gene expression level, the more sensitive the cell is tothe treatment). Eight genes, SRGN, COL6A3, GPSM3, HSD11B1, PEX6, RAC2,SSX5, and ACBD3, had expression levels that were negatively associatedthe IC₅₀ values (i.e., the higher the gene expression level, the moresensitive the cell is to the treatment; or the lower the gene expressionlevel, the more resistant the cell is to the treatment).

Example 4 Gene Signatures Associated with Compound a Sensitivity

These new groups of genes have been identified to be associated withcytotoxic sensitivity to Compound A after analyzing Compound A cytotoxicactivity data using a different gene expression profile database—CCLE(Cancer Cell Line Encyclopedia). Two independent data analysis methodswere used to identify gene signatures associated with Compound Asensitivity, i.e., the t-test and multi-regression methods.

In t-tests, the gene expression data were grouped from either sensitiveor resistant cells in the database and compared them to the general datapool, respectively, in order to identify the gene signature specificallyassociated with either a sensitive (group 1) or resistant (group 2)phenotype. In the multi-regression method, whole experimental IC50 datawas used to generate an IC50 prediction curve, which was comprised of 31genes (group 3) expression data using the multi-regression algorithm.The prediction curve will be used to predict the sensitivity (IC50) ofany given cells based on its gene expression data. Both methods havebeen popularly used to generate gene expression signatures and theresults from both methods for future experimental validation areincluded here.

Comparing with the previous gene list, there are two overlapped genes,NEDD4 and PAPSS2 (in (3) below).

-   -   (1) A gene expression signature comprised of these 28 genes has        been found by t-test in those cells which are sensitive to        Compound A:        TFP1, DDX60, MYL9, EREG, IL18, LOC253039, PLAU, CXCL2, FOXQ1,        PYCARD, TSTD1, AHNAK2, LHFP, LEPREL1, UCP2, IL1RAP, SNAI2, RHOF,        FZD5, PRTFDC1, MXRA7, STEAP2, C15orf48, SMARCA1, PTGES, EGFR,        SATB2, LOC100288092,    -   (2) A gene expression signature comprised of these 25 genes have        been found by t-test in those cells which are resistant to        Compound A:

EREG, GDPD5, IFITM1, LOC100129502, INHBB, S100A10, S100A6, SCML1, ABCC4,THRB, GNG12, SDCBP, PTGR1, GSTA4, MGST1, CELSR2, SEPT10, ARNTL2,SMARCA1, MSN, FAM43A, PROCR, ELOVL7, CAPG, ANXA1,

-   -   (3) A Compound A sensitivity prediction curve comprised of these        31 genes expression data using multi-regression method:        SMARCA1, CNKSR3, TFPI, FBXO17, NEDD4, ITGAV, COL18A1, WWTR1,        LOC100130776, HECTD2, SLC26A2, ADM, GJC1, RBPMS, PAPSS2, LAMB1,        TXNIP, FSCN1, ARHGDIB, TRAPPC2, DRAM1, CRIM1, NME4. UGDH,        FAM57A, AGPAT9, FMNL2, FAM114A1, GPR125, GALNT2, SLC7A11.

Example 5 Large-Scale Cancer Cell Line Screening with the SelectiveHDAC-6 Inhibitor Compound A Identifies Potential Cancer Targets andCandidate Predictive Biomarkers

Compound A is a selective inhibitor of HDAC6 (Histone Deacetylase 6)currently in Phase 1 clinical trials as a single agent and incombination with bortezomib (Velcade) or lenalidomide (Revlimid) inrelapsed and refractory multiple myeloma Inhibitors of HDACs have beenshown to exhibit potent anti-cancer activity mediated by increased levelof acetylation of both histone and non-histone proteins, resulting ingrowth arrest, cell differentiation and apoptosis. In the presentexample, the broader anti-tumor activity of Compound A was investigatedwith a panel of 65 human tumor cell lines derived from the major cancertypes to define additional oncology indications for potential clinicaldevelopment and to identify potential biomarkers of tumor sensitivityand drug resistance.

Among the tumor types examined, a number of lymphoma and leukemia celllines have shown substantial sensitivity to Compound A treatment. Twogene signatures have been identified for either sensitive or resistantcells, respectively, through the combination of cell viability,underlying genetics, and baseline gene expression data from cell linepanels and correlation of IC₅₀ values with gene expression levels asreported in the Cancer Cell Line Encyclopedia (CCLE). These geneticsignatures include genes in the ErbB signaling pathway, chromatinremodeling and protein ubiquitination. Biological pathway analysis usingthe gene signatures indicated an over-representation of certainsignaling pathways including the caspase cascade in apoptosis,PDGFR-alpha, S1P3, and ErbB4 signaling. Some of these results support aproposed cytotoxic activity of selective HDAC6 inhibition throughblocking of misfolded protein clearance, while other findings suggestfurther novel pathways that are impacted by the HDAC6 selectiveinhibitor Compound A.

Confirmation of predicted sensitive and resistant cell lines using thegene signatures will lead to the development of predictive biomarkersand selection of tumor type(s) for further in vitro and in vivovalidation with Compound A in combination with approved standard of caretherapeutics.

Example 6 Development of a 58 Gene Signature Prediction Model for BreastCancer

65 cancer cell lines were analyzed using the HDAC inhibitor Compound A.Lymphoma cells were identified as the most sensitive cell group toCompound A. A gene expression profile signature and several genemutations were demonstrated to be tightly associated with Compound Asensitivity, and can be used as biomarkers to predict a cancer'ssensitivity to the HDAC inhibitor Compound A.

Among the cancer cell groups that were studied, the breast cancer cellgroup comprised of 16 breast cancer cell lines was the only one thatproduced a confident 58 gene signature prediction model using acorrelation analysis of their IC50 values and their gene expression datapublished at the CCLE database (www dot broad institute dotorg/ccle/home). None of the other three other groups, lung cancer, AML,and lymphoma, were able to produce a confident model because ofeither: 1) the data set size, or 2) the narrow range of the IC50 values.

For the 58 genes in the “signature”, 35 low expression and 23 highexpression genes related to the “sensitive” signature, while 35 highexpression and 23 low expression related to the “resistant” signature(see FIG. 1). The names of the 58 genes are: transforming growth factorbeta-3 (TGFB3); CD44 molecule (Indian blood group) (CD44); cytochromep450, family 4, subfamily Z, polypeptide 2 pseudogene (CYP4Z2P);interferon-induced protein 44 (IFI44); solute carrier family 9,subfamily A (NHE6, cation proton antiporter 6), member 6 (SLC9A6);v-erb-b2 erythroblastic leukemia viral oncogene homolog 2,neuro/glioblastoma derived oncogene homolog (avian) (ERBB2); v-yes-1Yamaguchi sarcoma viral related oncogene homolog (LYN); pleckstrinhomology-like domain, family A, member 1 (PHLDA1); peroxisomeproliferator-activated receptor gamma (PPARG); dicarbonyl/L-xylulosereductase (DCXR); uridine phosphorylase 1 (UPP1); ATP-binding cassette,sub-family C (CFTR/MRP), member 11 (ABCC11); aldo-keto reductase family1, member C2 (dihydrodiol dehydrogenase 2; bile acid binding protein;3-alpha hydroxysteroid dehydrogenase, type III) (AKR1C2);BCL2-associated athanogene 2 (BAG2); TLR4 interactor with leucine-richrepeats (TRIL); uncharacterized LOC440335 (LOC440335); inhibin, beta B(INHBB); dickkopf 1 homolog (Xenopus laevis) (DKK1); insulin receptorsubstrate 2 (IRS2); chromosome 17 open reading frame 28 (C17orf28); LIMdomain kinase 2 (LIMK2); like-glycosyltransferase (LARGE); coiled-coildomain containing 82 (CCDC82); solute carrier family 40 (iron-regulatedtransporter), member 1 (SLC40A1); interferon-induced protein withtetratricopeptide repeats 1 (IFIT1); formin-like 2 (FMNL2); leukemiainhibitory factor (LIF); transforming growth factor, beta recetor 2(70/80 kDa) (TGFBR2); G protein-coupled receptor 160 (GPR160); cytokineinducible SH2-containing protein (CISH); phospholipase C, beta 4(PLCB4); B-cell linker (BLNK); phospholipase C, gamma 2(phosphatidylinositol-specific) (PLCG2); caveolin 2 (CAV2); prolinedehydrogenase (oxidase) 1 (PRODH); ras homolog family member B (RHOB);interferon-induced protein with tetratricopeptide repeats 3 (IFIT3);calbindin 2 (CALB2); TSPY-like 5 (TSPYL5); chromosome X open readingframe 61 (CXorf61); hematopoietically expressed homeobox (HHEX); cAMPresponsive element binding protein 3-like4 (CREB3L4); X-box bindingprotein 1 (XBP1); SAM pointed domain containing ets trsanscriptionfactor (SPDEF); nuclear receptor coactivator 7 (NCOA7); galaninprepropeptide (GAL); HECT and RLD domain containing E3 ubiquitin proteinligase 5 (HERC5); major histocompatibility complex, class I, A (HLA-A);centromere protein V (CENPV); frequently rearranged in advanced T-celllymphomas 2 (FRAT2); phospholipase B domain containing 1 (PLBD1);adenosine A2b receptor (ADORA2B); G protein-coupled receptor, family C,group 5, member A (GPRC5A); enoyl CoA hydratase domain containing 1(ECHDC1); guanylate binding protein 1, interferon-inducible (GBP1);sulfatase 2 (SULF2), uncharacterized LOC100507463 (LOC100507463), andKIAA1324 (KIAA1324).

Of the 58 genes above, the following high expression genes are relatedto the “sensitive” signature: TGFB3, CYP4Z2P, ERBB2, DCXR, ABCC11, TRIL,LOC440335, INHBB, C17orf28, LIMK2, LARGE, SLC40A1, GPR160, CISH, PLCB4,BLNK, PRODH, RHOB, CREB3L4, XBP1, SPDEF, FRAT2, and KIAA1324; and thefollowing low expression genes are related to the “sensitive” signature:CD44, IFI44, SLC9A6, LYN, PHLDA1, PPARG, UPP1, AKR1C2, BAG2, DKK1, IRS2,IFIT1, FMNL2, LIF, TGFBR2, PLCG2, CAV2, IFIT3, CALB2, TSPYL5, CXorf61,HHEX, NCOA7, GAL, HERC5, HLA-A, CENPV, PLBD1, ADORA2B, GPRC5A, ECHDC1,GBP1, SULF2, and LOC100507463.

Of the 58 genes above, the following low expression genes are related tothe “resistant” signature: TGFB3, CYP4Z2P, ERBB2, DCXR, ABCC11, TRIL,LOC440335, INHBB, C17orf28, LIMK2, LARGE, SLC40A1, GPR160, CISH, PLCB4,BLNK, PRODH, RHOB, CREB3L4, XBP1, SPDEF, FRAT2, and KIAA1324; and thefollowing high expression genes are related to the “resistant”signature: CD44, IFI44, SLC9A6, LYN, PHLDA1, PPARG, UPP1, AKR1C2, BAG2,DKK1, IRS2, IFIT1, FMNL2, LIF, TGFBR2, PLCG2, CAV2, IFIT3, CALB2,TSPYL5, CXorf61, HHEX, NCOA7, GAL, HERC5, HLA-A, CENPV, PLBD1, ADORA2B,GPRC5A, ECHDC1, GBP1, SULF2, and LOC100507463.

Compound A sensitivity of 59 breast cell lines was predicted based upontheir gene expression profile data published at the CCLE site using the58 gene signature (see FIG. 2). Two statistically well-supported groupswere identified. The first group (32 cell lines) was associated withsensitive cells, which were defined as having an IC50<5 μM. The secondgroup (27 cell lines) was associated with resistant cells, which weredefined as having an IC50>5 μM. In order to validate the Compound Asensitivity prediction power of the model, tested 4 new breast cancercell lines were tested, and their IC50 values fit well to theestablished model.

The same procedure above was applied to 352 primary breast tumor tissuesin the expO database (www dot intgen dot org/expo/). The sensitivity ofthese primary cells to Compound A was predicted by testing thecorrelation of their gene expression with the gene expression of the twoCCLE groups (sensitive and resistant) (see FIG. 2). Two stable consensusgroups were identified within the tested breast tumors (260 predictedsensitive tissues, 90 predicted resistant tissues, with 2 unclassified).One stable consensus group (260 predicted sensitive tissues) was foundto be similar to the sensitive CCLE breast cancer cell group, and theother stable consensus group (90 predicted resistant tissues) was foundto be similar to the resistant CCLE breast cancer cell group. Nosignificantly distinct groups were identified within either lung orcolon tumor tissues (see FIG. 3).

The sensitive group of breast tumor tissues was enriched for threeclinical breast cancer diagnosis markers: ER positive, PR positive, andlow grade breast tumors. The resistant group of breat tumor tissues wasassociated with a triple negative (ER−PR−Her2/neu−) marker. See FIG. 4.

In conclusion, a stable gene expression signature was identified thatcould predict Compound A sensitivity in breast cancer.

Example 7 Summary of the 16 Breast Cancer Cell Line IC50 Data

Table 1 shows a summary of the 16 breast cancer cell line IC50 data,which were used to model the predictive gene expression biomarkers(Note: Only 14 of these 16 were found in the CCLE data and used in themodeling).

Among many genetic markers of these cells, the ERBB2 (gene for Her2)amplification genotype was tightly associated with Compound Asensitivity phenotype (Table 1). Actually, ERBB2 is one of the 58 genesin the model itself. Although ESR1 (gene for ER) and PSR (gene for PR)were not picked in the top 58 gene list during the cell line dataanalysis, however, when using this 58-gene signature to analyze the 352clinical breast tumor data in expO data, both ER+ and PR+ weresignificantly associated with the “predicted sensitive” group.

TABLE 1 Compound A Cells ERBB2 IC₅₀ (μM) MDA-MB-453 Amplified 1.25SK-BR-3 Amplified 1.94 BT 474 Amplified 2.32 ZR-75-1 3.57 MDA-MB-361Amplified 4.51 T-47D* 5.00 Hs578T 5.57 MX-1 6.38 MDA-MB-231 7.49 Bcap-377.66 HCC38 7.76 MCF-7 8.95 BT-549 8.97 HCC1937 11.25 MDA-MB-468 14.92MDA-MB-436 25.66

Example 8 Synthesis of2-(diphenylamino)-N-(7-(hydroxyamino)-7-oxoheptyl)pyrimidine-5-carboxamide (Compound A)

Reaction Scheme

Synthesis of Intermediate 2

A mixture of aniline (3.7 g, 40 mmol), ethyl2-chloropyrimidine-5-carboxylate 1 (7.5 g, 40 mmol), K₂CO₃ (11 g, 80mmol) in DMF (100 ml) was degassed and stirred at 120° C. under N2overnight. The reaction mixture was cooled to rt and diluted with EtOAc(200 ml), then washed with saturated brine (200 ml×3). The organic layerwas separated and dried over Na₂SO₄, evaporated to dryness and purifiedby silica gel chromatography (petroleum ethers/EtOAc=10/1) to give thedesired product as a white solid (6.2 g, 64%).

Synthesis of Intermediate 3

A mixture of the compound 2 (6.2 g, 25 mmol), iodobenzene (6.12 g, 30mmol), CuI (955 mg, 5.0 mmol), Cs₂CO₃ (16.3 g, 50 mmol) in TEOS (200 ml)was degassed and purged with nitrogen. The resulting mixture was stirredat 140° C. for 14 h. After cooling to rt, the residue was diluted withEtOAc (200 ml) and 95% EtOH (200 ml), NH₄F—H₂O on silica gel [50 g,pre-prepared by the addition of NH₄F (100 g) in water (1500 ml) tosilica gel (500 g, 100-200 mesh)] was added, and the resulting mixturewas kept at rt for 2 h, the solidified materials was filtered and washedwith EtOAc. The filtrate was evaporated to dryness and the residue waspurified by silica gel chromatography (petroleum ethers/EtOAc=10/1) togive a yellow solid (3 g, 38%).

Synthesis of Intermediate 4

2N NaOH (200 ml) was added to a solution of the compound 3 (3.0 g, 9.4mmol) in EtOH (200 ml). The mixture was stirred at 60° C. for 30 min.After evaporation of the solvent, the solution was neutralized with 2NHCl to give a white precipitate. The suspension was extracted with EtOAc(2×200 ml), and the organic layer was separated, washed with water(2×100 ml), brine (2×100 ml), and dried over Na₂SO₄. Removal of solventgave a brown solid (2.5 g, 92%).

Synthesis of Intermediate 6

A mixture of compound 4 (2.5 g, 8.58 mmol), aminoheptanoate 5 (2.52 g,12.87 mmol), HATU (3.91 g, 10.30 mmol), DIPEA (4.43 g, 34.32 mmol) wasstirred at rt overnight. After the reaction mixture was filtered, thefiltrate was evaporated to dryness and the residue was purified bysilica gel chromatography (petroleum ethers/EtOAc=2/1) to give a brownsolid (2 g, 54%).

Synthesis of2-(diphenylamino)-N-(7-(hydroxyamino)-7-oxoheptyl)pyrimidine-5-carboxamide

A mixture of the compound 6 (2.0 g, 4.6 mmol), sodium hydroxide (2N, 20mL) in MeOH (50 ml) and DCM (25 ml) was stirred at 0° C. for 10 minHydroxylamine (50%) (10 ml) was cooled to 0° C. and added to themixture. The resulting mixture was stirred at rt for 20 min. Afterremoval of the solvent, the mixture was neutralized with 1M HCl to givea white precipitate. The crude product was filtered and purified bypre-HPLC to give a white solid (950 mg, 48%).

1. A method for predicting whether a cancer patient will respond totreatment with a histone deacetylase (HDAC) inhibitor comprising: a)determining whether the human epidermal growth factor receptor 2 (Her2)protein is overexpressed, as compared to a normalized protein expressionlevel of the protein, in a biological sample from a cancer patient; andeither correlating the presence of such overexpression as an indicationthat the patient will respond to such treatment, or correlating theabsence of such overexpression as an indication that the patient willnot respond to such treatment; or b) measuring an expression level of agene selected from the group consisting of pterin-4 alpha-carbinolaminedehydratase/dimerization cofactor of hepatocyte nuclear factor 1 alpha(PCBD1); protein phosphatase 2, regulatory subunit B, gamma isoform(PPP2R2C); neural precursor cell expressed, developmentallydownregulated 4 (NEDD4); prolyl 4-hydroxylase, alpha polypeptide II(P4HA2); SLC2A4 regulator (SLC2A4RG); sulfatase 2 (SULF2); lysosomalprotein transmembrane 4 alpha (LAPTM4A); 3′-phosphoadenosine5′-phosphosulfate synthase 2 (PAPSS2); aldo-keto reductase family 1,member C1 (dihydrodiol dehydrogenase 1; 20-alpha(3-alpha)-hydroxysteroid dehydrogenase) (AKR1C1); protein tyrosinephosphatase, non-receptor type 12 (PTPN12); DCN1, defective in cullinneddylation 1, domain containing 4 (S. cerevisiae) (DCUN1D4);ras-related C3 botulinum toxin substrate 2 (RAC2); acyl-Coexzyme Adehydrogenase, C-4 to C-112 straight chain (ACADM); Rho GTPaseactivating protein 4 (ARHGAP4); ATPase type 13A1 (ATP13A1); chemokinereceptor 7 (CCR7); coronin 7 (CORO7); CXXC finger 4 (CXXC4);differentially expressed in FDCP 6 homolog (DEF6); KRI1 homolog (KRI1);limb region 1 homolog (LMBR1L); leukotriene B4 receptor (LTB4R);RAD54-like 2 (RAD54L2); chromosome X open reading frame 21 (CXorf21);SREBF chaperone (SCAP); selectin L (SELL); splicing factor 3a, subunit 2(SF3A2); Lyrm7 homolog (LYRM7); O-linked N-acetylglucosamine transferase(OGT); tubulin, alpha 3c (TUBA3C); tubulin, alpha 3d (TUBA3D); KH-typesplicing regulatory protein (KHSRP); DEAH (Asp-Glu-Ala-His) boxpolypeptide 30 (DHX30); APEX nuclease (apurinic/apyrimidinicendonuclease) 2 (APEX2); and abhydrolase domain containing 14A (ABHD14A)in a biological sample from the cancer patient; and either correlating alow expression level of any one or more of the genes PCBD1, PPP2R2C,NEDD4, P4HA2, SLC2A4RG, SULF2, LAPTM4A, PAPSS2, AKR1C1, PTPN12, andDCUN1D4, as compared to a normalized gene expression level, as anindication that the patient will respond to such treatment; orcorrelating a high expression level of any one or more of the genesRAC2, ACADM, ARHGAP4, ATP13A1, CCR7, CORO7, CXXC4, DEF6, KRI1, LMBR1L,LTB4R, RAD54L2, CXorf21, SCAP, SELL, SF3A2, LYRM7, OGT, TUBA3C, TUBA3D,KHSRP, DHX30, APEX2, and ABHD14A, as compared to a normalized geneexpression level, as an indication that the patient will respond to suchtreatment.
 2. A method for predicting whether a cancer patient willrespond to combination treatment with a histone deacetylase (HDAC)inhibitor and a proteasome inhibitor comprising: a) determining whethera gene mutation in the SMAD family member 4 (SMAD4) gene is present in abiological sample from a cancer patient; and either correlating thepresence of a gene mutation as an indication that the patient willrespond to such treatment, or correlating the absence of a gene mutationas an indication that the patient will not respond to such treatment; orb) determining whether a gene mutation in a first gene selected from thegroup consisting of phosphatase and tensin homolog (PTEN), epidermalgrowth factor receptor oncogene (EGFR), histone-lysineN-methyltransferase (EZH2), SET domain containing 2 (SETD2), and vonHippel-Lindau tumor suppressor (VHL), is present in a biological samplefrom the cancer patient and either correlating the presence of one ormore such mutations as an indication that the patient will respond tosuch treatment or correlating the absence of such mutations as anindication that the patient will not respond to such treatment; or c)measuring the expression level of a second gene selected from the groupconsisting of UDP-glucose dehydrogenase (UGDH); H2A histone family,member Y2 (H2AFY2); myosin VC (MYO5C); nephronectin (NPNT); KIAA1598(KIAA1598); serglycin (SRGN); collagen, type VI, alpha 3 (COL6A3):G-protein sing modulator 3 (GPSM3): hydroxysteroid dehydrogenase 1(HSD11B1): peroxisomal biogenesis factor 6 (PEX6): ras-related C3botulinum toxin substrate 2 (RAC2); synovial sarcoma, X breakpoint 5(SSX5); and acyl-Coenzyme A binding domain containing 3 (ACBD3) in abiological sample from the cancer patient and either correlating a lowexpression level of any one or more of the genes UGDH, H2AFY2, MYO5C,NPNT, and KIAA1598, as compared to a normalized gene expression level,as an indication that the patient will respond to such treatment; orcorrelating a high expression level of any one or more of the genesSRGN, COL6A3, GPSM3, HSD11B1, PEX6, RAC2, SSX5, and ACBD3, as comparedto a normalized gene expression level, as an indication that the patientwill respond to such treatment. 3-6. (canceled)
 7. The method of claim2, wherein the cancer is selected from the group consisting of:brain/neuronal cancer, breast cancer, cancer of the central nervoussystem, haematopoietic and lymphoid tissue cancer, kidney cancer, cancerof the large intestine, liver cancer, lung cancer, cancer of theoesophagus, pancreatic cancer, prostate cancer, skin cancer, soft tissuecancer, and stomach cancer.
 8. The method of claim 2, wherein the HDACinhibitor is a HDAC6 inhibitor.
 9. The method of claim 8, wherein theHDAC6 inhibitor is a compound of formula I:

or a pharmaceutically acceptable salt, ester or prodrug thereof. 10.(canceled)
 11. The method of claim 2, wherein the proteasome inhibitoris bortezomib.
 12. (canceled)
 13. The method of claim 1, furthercomprising the step of administering to the patient a therapeuticallyeffective amount of a HDAC inhibitor.
 14. The method of claim 2, furthercomprising the step of administering to the patient a therapeuticallyeffective amount of a HDAC inhibitor and a proteasome inhibitor. 15.(canceled)
 16. A method for predicting whether a breast cancer patientwill respond to treatment with a histone deacetylase (HDAC) inhibitorcomprising the steps of: a) measuring the expression level of each ofthe following genes: transforming growth factor beta-3 (TGFB3); CD44molecule (Indian blood group) (CD44); cytochrome p450, family 4,subfamily Z, polypeptide 2 pseudogene (CYP4Z2P); interferon-inducedprotein 44 (IFI44); solute carrier family 9, subfamily A (NHE6, cationproton antiporter 6), member 6 (SLC9A6); v-erb-b2 erythroblasticleukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogenehomolog (avian) (ERBB2); v-yes-1 Yamaguchi sarcoma viral relatedoncogene homolog (LYN); pleckstrin homology-like domain, family A,member 1 (PHLDA1); peroxisome proliferator-activated receptor gamma(PPARG); dicarbonyl/L-xylulose reductase (DCXR); uridine phosphorylase 1(UPP1); ATP-binding cassette, sub-family C (CFTR/MRP), member 11(ABCC11); aldo-keto reductase family 1, member C2 (dihydrodioldehydrogenase 2; bile acid binding protein; 3-alpha hydroxysteroiddehydrogenase, type III) (AKR1C2); BCL2-associated athanogene 2 (BAG2);TLR4 interactor with leucine-rich repeats (TRIL); uncharacterizedLOC440335 (LOC440335); inhibin, beta B (INHBB); dickkopf 1 homolog(Xenopus laevis) (DKK1); insulin receptor substrate 2 (IRS2); chromosome17 open reading frame 28 (C17orf28); LIM domain kinase 2 (LIMK2);like-glycosyltransferase (LARGE); coiled-coil domain containing 82(CCDC82); solute carrier family 40 (iron-regulated transporter), member1 (SLC40A1); interferon-induced protein with tetratricopeptide repeats 1(IFIT1); formin-like 2 (FMNL2); leukemia inhibitory factor (LIF);transforming growth factor, beta recetor 2 (70/80 kDa) (TGFBR2); Gprotein-coupled receptor 160 (GPR160); cytokine inducible SH2-containingprotein (CISH); phospholipase C, beta 4 (PLCB4); B-cell linker (BLNK);phospholipase C, gamma 2 (phosphatidylinositol-specific) (PLCG2);caveolin 2 (CAV2); proline dehydrogenase (oxidase) 1 (PRODH); rashomolog family member B (RHOB); interferon-induced protein withtetratricopeptide repeats 3 (IFIT3); calbindin 2 (CALB2); TSPY-like 5(TSPYL5); chromosome X open reading frame 61 (CXorf61);hematopoietically expressed homeobox (HHEX); cAMP responsive elementbinding protein 3-like4 (CREB3L4); X-box binding protein 1 (XBP1); SAMpointed domain containing ets trsanscription factor (SPDEF); nuclearreceptor coactivator 7 (NCOA7); galanin prepropeptide (GAL); HECT andRLD domain containing E3 ubiquitin protein ligase 5 (HERC5); majorhistocompatibility complex, class I, A (HLA-A); centromere protein V(CENPV); frequently rearranged in advanced T-cell lymphomas 2 (FRAT2);phospholipase B domain containing 1 (PLBD1); adenosine A2b receptor(ADORA2B); G protein-coupled receptor, family C, group 5, member A(GPRC5A); enoyl CoA hydratase domain containing 1 (ECHDC1); guanylatebinding protein 1, interferon-inducible (GBP1); sulfatase 2 (SULF2),uncharacterized LOC100507463 (LOC100507463), and KIAA1324 (KIAA1324) ina biological sample from the breast cancer patient; and b) correlating ahigh expression level, as compared to a normalized gene expression levelof the gene, of the following genes TGFB3, CYP4Z2P, ERBB2, DCXR, ABCC11,TRIL, LOC440335, INHBB, C17orf28, LIMK2, LARGE, SLC40A1, GPR160, CISH,PLCB4, BLNK, PRODH, RHOB, CREB3L4, XBP1, SPDEF, FRAT2, and KIAA1324 asan indication that the patient will respond to such treatment; andcorrelating a low expression level, as compared to a normalized geneexpression level of the gene, of the following genes CD44, IFI44,SLC9A6, LYN, PHLDA1, PPARG, UPP1, AKR1C2, BAG2, DKK1, IRS2, IFIT1,FMNL2, LIF, TGFBR2, PLCG2, CAV2, IFIT3, CALB2, TSPYL5, CXorf61, HHEX,NCOA7, GAL, HERC5, HLA-A, CENPV, PLBD1, ADORA2B, GPRC5A, ECHDC1, GBP1,SULF2, and LOC100507463 as an indication that the patient will respondto such treatment.
 17. The method of claim 16, wherein the methodfurther comprises the step of administering to the patient atherapeutically effective amount of a HDAC inhibitor.
 18. The method ofclaim 1, wherein the cancer is selected from the group consisting of:brain/neuronal cancer, breast cancer, cancer of the central nervoussystem, haematopoietic and lymphoid tissue cancer, kidney cancer, cancerof the large intestine, liver cancer, lung cancer, cancer of theoesophagus, pancreatic cancer, prostate cancer, skin cancer, soft tissuecancer, and stomach cancer.
 19. The method of claim 1, wherein the HDACinhibitor is a HDAC6 inhibitor.
 20. The method of claim 19, wherein theHDAC6 inhibitor is a compound of formula I:

or a pharmaceutically acceptable salt, ester or prodrug thereof.